https://orcid.org/0000-0002-3374-1038
Figures
Abstract
Varicella-zoster virus (VZV), the cause of shingles, remains a significant health issue worldwide, particularly among aging populations. The recombinant zoster vaccine (Shingrix, RZV) is the only approved vaccine in many countries and has demonstrated >90% efficacy with durable protection lasting over a decade, highlighting the value of subunit vaccines targeting VZV glycoprotein E (VZV gE). Although RZV provides durable and highly effective protection, alternative vaccine platforms remain important for advancing antigen design and improving immune presentation. Advances in nanoparticle technology now enable antigens to be displayed in highly ordered, repetitive arrays, offering new opportunities to strengthen antiviral immunity. Here, we developed a novel nano-vaccine named Nano-gEVZV, which employs the antigen from the licensed Hepatitis E vaccine (Hecolin) as a nanoparticle scaffold and uses a nano-binder (NB) to display VZV gE in a repetitive arrangement. Nano-gEVZV demonstrates enhanced antigen uptake by antigen-presenting cells (APCs), improved lymph node retention, and stronger B- and T-cell responses compared with Shingrix in mouse models. While RZV remains the gold standard for herpes zoster prevention, we explored a nanoparticle-based gE display platform as a complementary approach to improve manufacturability and immune presentation.
Author summary
Shingles, caused by reactivation of the VZV, remains a major health concern in older adults, for whom vaccination is the most effective preventive measure. In this study, we designed a nanoparticle-based gE vaccine (Nano-gEVZV) using a hepatitis E vaccine–derived platform to explore strategies for enhancing shingles vaccine immunogenicity. The Nano-gEVZV formulation, combined with an AS01B-like adjuvant, promoted efficient lymph node localization and enhanced antigen uptake by dendritic cells. In mouse models, Nano-gEVZV elicited stronger VZV gE-specific humoral and CD4 ⁺ T-cell responses compared with monomeric antigen formulations, highlighting how nanoparticle presentation can synergize with adjuvants to shape adaptive immunity. While these findings provide proof-of-concept evidence for the potential of modular nanoparticle display in herpes zoster vaccine development, further validation in human-relevant systems will be essential before clinical translation.
Citation: Xue W, Wang H, Zeng Y, Zhang S, Cui L, Liu H, et al. (2026) A modular nanoparticle display strategy for varicella-zoster virus gE based on a licensed protein scaffold. PLoS Pathog 22(3): e1013983. https://doi.org/10.1371/journal.ppat.1013983
Editor: Eain A. Murphy, State University of New York Upstate Medical University, UNITED STATES OF AMERICA
Received: October 30, 2025; Accepted: February 6, 2026; Published: March 2, 2026
Copyright: © 2026 Xue et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data generated or analyzed during this study are included in this manuscript and supporting information.
Funding: This work was supported by grants from the National Key Research and Development Program of China (grant no. 2023YFC2307602 to Jun Zhang), the National Natural Science Foundation of China (grant no. 82272310 to Tong Cheng, 82471861 to T.L.), Natural Science Foundation of Xiamen (grant no. 3502Z20227165 to H.Y.), Fundamental Research Funds for the Central Universities (grant nos. 20720220004 to S.L., 20720220006 to N.X.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests. S.L., T.L., W.X., Y. Gu, J.Z. and N.X. are inventors on a patent application (PCT/CN2023/129973) filed by the Xiamen University that covers nanobody mediating the p239 particle decoration described in this work. The authors declare no other competing financial interests.
Introduction
The Varicella Zoster Virus (VZV) is a highly contagious neurotropic α-herpesvirus that exclusively infects humans [1]. Primary infection usually occurs in childhood, manifesting as chickenpox [2]. With weakened immunity due to aging or immunosuppression, the virus may reactivate from its latent state, leading to herpes zoster (shingles) [3]. This reactivation is frequently associated with severe complications, including postherpetic neuralgia (PHN), and represents a considerable health threat, particularly to the elderly [4]. Consequently, herpes zoster (HZ) significantly impairs the quality of life for those afflicted and has become one of the most pressing global health concerns [5].
Vaccination remains the cornerstone of infectious disease control, and the development of effective vaccines is paramount in preventing herpes zoster [6]. Zostavax (zoster vaccine live, or ZVL), an attenuated live vaccine approved by the FDA in 2006, provided the first preventive option but showed declining effectiveness with increasing age [7,8]. In contrast, the recombinant subunit vaccine Shingrix (RZV), licensed in 2017, represents a major advance in herpes zoster prevention. By combining the essential VZV gE antigen with the potent AS01B adjuvant system, Shingrix achieves over 90% protection across age groups, including the elderly, and offers durable efficacy extending for at least a decade. Furthermore, it has demonstrated substantial effectiveness in immunocompromised individuals, a population at elevated risk of severe disease. [9,10]. These achievements have established Shingrix as the current global standard of care and a milestone in vaccinology, underscoring the central role of subunit vaccine and CD4 ⁺ T-cell responses in HZ protection.
Building on this success, alternative vaccine platforms are being actively explored to advance antigen design and to better understand how antigen organization influences immune responses. Particulate antigen formulations—including protein nanoparticles and virus-like particles (VLPs) such as ferritin [11], I53-50 [12,13] and Mi3 [14] have shown the ability to present antigens in multivalent, geometrically ordered arrays. These structures enhance lymph node trafficking, promote uptake by antigen-presenting cells, and facilitate MHC II–mediated CD4 ⁺ T-cell priming, leading to augmented type 1 helper T-cell (Th1) and T follicular helper (Tfh) responses, and durable humoral immunity [15–17]. Notably, Li et al., demonstrated that VZV gE displayed on multiple nanoparticle platforms elicited stronger humoral and cellular immunity in naïve mice, with one construct (gEM) showing superior CD4 ⁺ T-cell responses in both VZV-primed mice and rhesus macaques [18]. Their findings highlight that nanoparticle-based presentation of VZV gE can drive potent and durable cell-mediated immunity, underscoring the promise of nanoparticle technology as a next-generation strategy for herpes zoster prevention.
In this study, we extend these advances by implementing a nanobody-mediated modular anchoring strategy to display VZV gE in a dense, oriented array on the p239 scaffold [19] (from the licensed HEV vaccine Hecolin [20,21]). We performed detailed structural and functional analyses of this construct, termed Nano-gEVZV, and evaluated its capacity for antigen uptake, lymph node retention, and induction of B-cell and CD4 ⁺ T-cell responses in comparison to RZV within an AS01B-like adjuvant (XUA01) milieu. Collectively, these findings demonstrate the potential of Nano-gEVZV as a next-generation vaccine candidate and provide translational insights into pairing particulate antigens with milder adjuvants for herpes zoster prevention.
Result
Construction and characterization of nano-gEVZV
The envelope glycoprotein E (gE) of VZV is critical for eliciting protective immunity. To construct a nanoparticle vaccine candidate, we employed the hepatitis E vaccine antigen p239 (derived from the licensed Hecolin vaccine) as a self-assembling nanoparticle scaffold for surface antigen display. However, because of the unique asymmetric structure of p239, direct surface fusion of heterologous proteins—using conventional fusion or SpyTag/SpyCatcher systems—is inefficient. To overcome this limitation, we developed a novel strategy that utilizes high-affinity nanobodies, isolated from an alpaca immune library, as nano-binders (NBs) to mediate specific anchoring of VZV gE on the nanoparticle surface (Fig 1A).
(A) The amino acid sequence and a diagram of Nano-gEVZV. (B) SDS-PAGE and corresponding immunoblot analysis of candidate fusion proteins. (C) Results of affinity analysis of nano-binders. P28C demonstrates the highest affinity. (D) The antigenicity of P28C-gEVZV characterized using an antibody array. (E) Purification of Nano-gEVZV using size exclusion chromatography. (F) Purified Nano-gEVZV were analyzed by gradient SDS-PAGE. (G) Sizes of Nano-gEVZV (purple) and the particle carrier (yellow) were analyzed using dynamic light scattering (DLS). (H)Transmission electron microscopical images of negatively stained p239 (left) and Nano-gEVZV (right). The illustrations in this figure were created using Figdraw: https://www.figdraw.com.
Six candidate NBs were obtained and genetically fused to the N-terminus of VZV gE (S1 Table). The resulting fusion proteins were expressed in insect cells and purified using nickel (Ni) affinity chromatography. SDS-PAGE analysis showed distinct bands at approximately 80 kDa, consistent with the expected size. A VZV gE-specific linear epitope antibody (1B11) can accurately detect the target protein (Fig 1B). Surface plasmon resonance (SPR) assays revealed that the NB P28C displayed the highest binding affinity (KD = 2.42 nM) for the p239 carrier among the six candidates (S1 Fig). Therefore, P28C was selected for nanoparticle assembly (Fig 1C).
To generate the final vaccine construct, P28C was fused to the N-terminus of VZV gE, producing the P28C-gEVZV fusion protein. In this configuration, P28C specifically binds to p239, directing the surface display of VZV gE and enabling ordered multivalent antigen presentation. Antigenicity profiling showed strong recognition of P28C-gEVZV by a panel of anti-VZV gE antibodies, indicating that the epitope conformation was well preserved (Fig 1D). For nanoparticle assembly, purified P28C-gEVZV was mixed with p239 at a molar ratio of 1:2. Saturation binding of VZV gE was confirmed by the chromatography profile, which exhibited a characteristic peak shift and a significant increase in absorbance at 280 nm (Fig 1E). SDS-PAGE of the main elution fraction showed two distinct components corresponding to the scaffold and fusion protein (Fig 1F). Dynamic light scattering (DLS) analyses show that the hydrodynamic radius increased from 14.2 nm (p239 alone) to 38.1 nm for Nano-gEVZV (Fig 1G). Transmission electron microscopy (TEM) visualized uniform, spherical particles consistent with virus-like morphology (Fig 1H). Collectively, these results confirm the successful construction of the Nano-gEVZV vaccine, demonstrating that the nanobody-mediated strategy effectively enables stable, multivalent surface display of VZV gE on a licensed nanoparticle scaffold.
Nano-gEVZV migrate to lymph node and uptake by DC2.4 cells
Lymph nodes play an essential role in adaptive immune responses. To monitor the in vivo distribution of Nano-gEVZV, we labeled Nano-gEVZV and the P28C-gEVZV with the red fluorescent dye (Alexa Fluor 647) and injected them subcutaneously into the footpads of mice. Antigen distribution was monitored at multiple time points using an IVIS Spectrum imaging system. By day 2 post-injection, a faint Nano-gEVZV signal was detected in both the inguinal and popliteal lymph nodes. This signal progressively intensified over time, reaching its peak on the fifth day (Figs 2A–2C and S2). Conversely, the signal from the P28C-gEVZV was mainly confined to the inguinal lymph nodes and diminished rapidly after peaking at day 3. These results indicate that Nano-gEVZV can rapidly drain to lymph nodes and remain there for an extended period, which is crucial for initiating an efficient immune response.
(A) In vivo fluorescence imaging of Alexa Fluor 647-labeled Nano-gEVZV after subcutaneous injection into the mouse footpad. The inguinal and popliteal lymph nodes (LN) are indicated by red arrows. Sequential imaging shows the trafficking and accumulation of Nano-gEVZV in draining lymph nodes over time. (B) IVIS Spectrum imaging of the inguinal LN and popliteal LN after dissection. (C) Quantitative analysis of fluorescence signals of inguinal lymph nodes ex vivo (D) Quantitative analysis of the integrated intensity of fluorescence signals within DC2.4 cells. Data are presented as mean ± SD. (E) The internalization efficiency of Nano-gEVZV by DC2.4 cells in vitro analyzed using confocal microscopy. Nano-gEVZV is labeled in red, cell membranes are displayed in green, and cell nuclei are labeled in blue. Data are presented as mean ± SD (n = 3). (F) Flow cytometric analysis of antigen uptake by bone marrow-derived cells isolated from C57BL/6 mice following incubation with AF647-labeled Nano-gEVZV or P28C-gEVZV (G) Quantification of AF647 ⁺ cells showing a significantly higher proportion of Nano-gEVZV –positive cells compared with P28C-gEVZV. Data are presented as mean ± SD (n = 3). Statistical significance was determined using an unpaired two-tailed Student’s t test; P < 0.05 was considered statistically significant.
To evaluate antigen uptake by antigen-presenting cells (APCs), we assessed the internalization of Nano-gEVZV by DC2.4 cells using immunofluorescence microscopy. Cell membranes were stained with Dio dye to delineate cellular boundaries. Confocal imaging revealed markedly stronger intracellular fluorescence in DC2.4 cells incubated with Nano-gEVZV compared with those exposed to P28C-gEVZV (Figs 2D, 2E and S3B), indicating enhanced cellular uptake of the nanoparticle formulation. To validate these observations in a more physiological context, we further examined uptake by primary bone marrow–derived cells from C57BL/6 mice. Consistent with the cell line data, these primary cells exhibited significantly greater internalization of Nano-gEVZV than P28C-gEVZV (p < 0.0027), confirming that the particulate architecture intrinsically promotes antigen capture and processing (Figs 2F, 2G and S3C). Together, these results demonstrate that Nano-gEVZV not only drains efficiently to lymph nodes but is also preferentially internalized by dendritic cells, thereby facilitating antigen presentation and the induction of robust adaptive immune responses.
Immunogenicity of Nano-gEVZV in mice
To assess the immunogenicity, C57BL/6 mice were administered Nano-gEVZV or P28C-gEVZV, both formulated in an AS01B-like adjuvant (S4 Fig), at weeks 0 and 4 (Fig 3A). The study also included a comparative analysis with the commercially available subunit vaccines, Shingrix and Hecolin. To accurately control the immunization dosage, the content of VZV gE was quantified. Results from the double antibody sandwich assay revealed that VZV gE constituted 67% of the Nano-gEVZV composition. (Fig 3B), corroborated by SDS-PAGE analysis (S3A Fig). Thus, immunization doses were standardized to 5 µg/dose for both the P28C-gEVZV and Shingrix, and adjusted to 7.5 µg/dose for Nano-gEVZV (equivalent to 5 µg VZV gE).
(A) Immunization scheme and sample collection schedule in C57BL/6 mice (n = 5). (B) The proportion of VZV gE in the nanoparticle was determined by sandwich enzyme-linked immunosorbent assay (ELISA). (C) gE-specific IgG antibody titers after two rounds of immunization. (D-E) gE-specific IgG1 and IgG2c titers of antisera. (F) The p239-specific antibody titer determined by endpoint dilution assay. (G) Plaque reduction neutralization assay showing neutralizing activity against v-Oka. Data are presented as mean ± standard deviation (SD) from independent experiments. (H) Neutralizing antibody titers determined by plaque reduction neutralization assay after booster immunization (n = 5). One-way ANOVA with Tukey’s multiple comparisons test was used for inter-group statistical comparisons. P < 0.05 was considered statistically significant. The illustrations in this figure were created using Figdraw: https://www.figdraw.com.
The gE-specific antibody titers were determined using the endpoint ELISA method. Following the booster immunization, Nano-gEVZV induced a relatively high titer of gE-specific antibodies, approximately 6.5 log units, outperforming both P28C-gEVZV and the Shingrix vaccine by approximately 3-fold and 1.9-fold, respectively (Fig 3C). Furthermore, Nano-gEVZV elicited significantly elevated titers of both IgG1 (P = 0.0007 vs. P28C-gEVZV; P = 0.0014 vs. Shingrix) and IgG2c (P < 0.0048 vs. Shingrix), particularly highlighting a marked enhancement of IgG2c antibodies indicative of a stronger Th1-type response (Fig 3D and 3E). Notably, we also identified specific antibodies targeting the carrier (p239), with antibody titers reaching levels comparable to those induced by Hecolin (P > 0.9999). This finding indicates that Nano-gEVZV could potentially serve as a combined vaccine platform conferring protection against both VZV and HEV (Fig 3F).
To analyze neutralization activity, a plaque reduction neutralization test (PRNT) was conducted, with the effective titer defined as the concentration corresponding to a 50% inhibition. We found that antisera from Nano-gEVZV effectively protected ARPE-19 cells from v-Oka virus infection, as evidenced by plaque assay images (Figs 3G and S5). Quantitative analysis revealed that Nano-gEVZV induced significantly higher neutralizing antibody titers than both P28C-gEVZV (P < 0.0001) and Shingrix (P < 0.0001) (Fig 3H). Collectively, these results demonstrate that Nano-gEVZV markedly enhances both binding and neutralizing antibody responses compared with P28C-gEVZV or commercial vaccine formulations, underscoring its potential as a next-generation gE-based subunit vaccine platform.
Nano-gEVZV induces a strong cellular immune response in mice
Considering the pivotal role of T cells in controlling herpes zoster virus reactivation, we conducted a comprehensive evaluation of the cellular immune response elicited by Nano-gEVZV in comparison to other vaccination approaches. We began by examining alterations in T cell compartments across different groups through cytokine staining. This involved stimulating single splenocytes with 15-mer peptides from VZV gE that span the complete gE protein sequence with an 11-amino-acid overlap. Utilizing flow cytometry, we measured the frequencies of CD4⁺ and CD8 ⁺ T cells producing gE-specific cytokines (IFN-γ and IL-2). Notably, Nano-gEVZV markedly improved the IFN-γ–secreting CD4 ⁺ T cell response, showing significantly higher frequencies compared with both P28C-gEVZV (P < 0.0001) and the Shingrix vaccine (P = 0.0128) (Fig 4A and 4B). This enhancement in Th1-type CD4 ⁺ T cells is particularly important, as IFN-γ production is a key correlate of protection against herpes zoster [22,23]. However, the enhancement in the IFN-γ–secreting CD8 ⁺ T cell response was not significant compare to Shingrix (P = 0.1685) (Figs 4C and S6C). Regarding IL-2 responses, Nano-gEVZV elicited comparable levels of IL-2–secreting CD4 ⁺ T cells relative to Shingrix (P = 0.6905) and CD8 ⁺ T cells (P = 0.1280) (Fig 4D and 4E), indicating that both formulations induce similar levels of T cell activation through this pathway.
(A) Representative flow cytometry images following treatment with four vaccine modalities. (B-E) Positive cytokine production by CD4⁺ and CD8 ⁺ cells in response to VZV gE stimulation. Histograms comparing the frequencies of IFN-γ–positive CD4 ⁺ cells (B), IFN-γ–positive CD8 ⁺ cells (C), IL-2–positive CD4 ⁺ cells (D), and IL-2–positive CD8 ⁺ cells (E). (F) Representative ELISPOT images obtained with four vaccine modalities. (G-H) Quantitative measurements of the number of IFN-γ–secreting cells (G) and IL-2–secreting cells (H), respectively. Values are shown as mean ± SD (n = 5). One-way ANOVA with Tukey’s multiple comparisons test was used for inter-group statistical comparisons. P < 0.05 was considered statistically significant.
Consistent with the flow cytometry data, ELISpot assays confirmed a significant increase in the number of cells secreting IFN-γ (P < 0.0001) and IL-2 (P < 0.0001) in Nano-gEVZV-immunized mice compared with the Shingrix group (Figs 4F–4H and S7). Although both assays detected IL-2–producing T cells, ICS and ELISpot exhibited different trends in IL-2 ⁺ CD4 ⁺ T-cell responses. ICS analysis showed comparable IL-2 ⁺ CD4 ⁺ T-cell frequencies among groups, whereas ELISpot revealed a significantly higher number of IL-2–secreting cells in Nano-gEVZV–immunized mice. This discrepancy likely reflects methodological differences between the two assays: ICS measures intracellular cytokine expression in individual cells, while ELISpot quantifies overall cytokine secretion from a larger and potentially more diverse cell population, capturing low-frequency or polyfunctional responses that ICS may underestimate. Collectively, these data demonstrate that Nano-gEVZV significantly enhances cellular immune responses—especially IFN-γ–producing CD4 ⁺ T cells (P < 0.0001)—highlighting the potential of nanoparticle antigen design to improve the immunogenicity of subunit vaccines against herpes zoster.
Discussion
Herpes Zoster (HZ) represents a significant public health challenge worldwide, exacerbated by the rapid aging of global populations [24]. Advances in vaccine technology have led to successive breakthroughs in the prevention of HZ, evolving from early attenuated live vaccines to the more recently favored recombinant protein vaccines [25]. These advancements have significantly decreased the incidence of HZ [26]. Importantly, the success of Shingrix has demonstrated that strong CD4 ⁺ T cell responses are central to durable protection against herpes zoster, establishing a benchmark for future vaccine development [27,28]. Accordingly, strategies that can further augment antigen presentation and CD4 ⁺ T cell priming are of particular interest in next-generation vaccine design [29,30]. Previous studies have demonstrated that nanoparticle-based presentation of VZV gE can substantially enhance immunogenicity across multiple platforms [18]. Consistent with and extending these findings, our study introduces an NB-mediated modular display to anchor VZV gE at high density on p239, paired with an AS01B-like adjuvant (XUA01). Our research demonstrates that cell-mediated immune responses are closely linked to the morphology of the immunogens. Replacing monomeric immunogens with nanoparticles substantially boosts both humoral and cell-mediated immune responses in mice, with pronounced effects on CD4 ⁺ T cell responses. In contrast to the computationally designed nanoparticle platform reported by Li et al. (2024), the Nano-gEVZV approach leverages a clinically validated scaffold together with a modular nanobody-based binding system, enabling programmable antigen display without de novo nanoparticle engineering. This strategy emphasizes structural adaptability and conceptual translatability, offering a complementary pathway for nanoparticle vaccine design.
The enhanced CD4 ⁺ T-cell immunity elicited by Nano-gEVZV can be explained by several complementary immunological mechanisms. First, the nanoparticle architecture enables more efficient uptake by antigen-presenting cells, promoting antigen processing through the MHC class II pathway and subsequent CD4 ⁺ T-cell activation [31,32]. Second, co-delivery of the nanoparticle antigen with a potent AS01B-like adjuvant supports innate immune activation and establishing a cytokine milieu that favors Th1 differentiation—an immune profile considered essential for protection against herpes zoster [33,34] (Fig 5). Although both monomeric and nanoparticle forms of VZV gE are ultimately processed into MHC-II–bound peptides, the superior CD4 ⁺ T-cell responses induced by Nano-gEVZV are most likely attributable to more efficient antigen uptake by antigen-presenting cells and subsequent cellular trafficking to the draining lymph nodes, thereby enhancing T-cell priming efficiency. Importantly, these findings indicate that antigen particleization does not substitute for potent adjuvants such as AS01B, but rather complements their effects by amplifying CD4 ⁺ T-cell activation. Our results therefore align with, rather than contradict, the immune mechanisms established for RZV, representing a conceptual advance in antigen design rather than a direct efficacy comparison.
Nanoparticle formulation promotes efficient antigen uptake, processing, and presentation by antigen-presenting cells, supports antigen retention within draining lymph nodes through APC-mediated transport, and ultimately enhances the magnitude and quality of adaptive immune responses. The illustrations in this figure were created using Figdraw: https://www.figdraw.com.
AS01B has demonstrated a strong safety profile and has been successfully incorporated into multiple licensed vaccines, including Recombinant Zoster Vaccine (RZV, Shingrix) [35], the RTS,S/AS01 malaria vaccine (RTS,S, Mosquirix) [36,37], and the Respiratory Syncytial Virus Prefusion F Protein vaccine (RSVPreF3, Arexvy) [38]. Although transient reactogenicity is relatively common, serious adverse events such as Guillain–Barré syndrome occur at extremely low rates (approximately 3 cases per million doses) comparable to other approved vaccines. Therefore, the development of alternative adjuvant systems should focus primarily on improving tolerability without compromising immunogenic potency rather than safety. Based on this rationale, pairing Nano-gEVZV with adjuvants that offer a more favorable reactogenicity profile may provide new opportunities for balancing tolerability and efficacy in herpes zoster vaccine design. Despite these advances, the translational scope of the present study remains preliminary. All immunogenicity data were obtained in C57BL/6 mice, which provide immunological consistency but cannot fully reflect the genetic and immunological diversity of human populations. We acknowledge that this limitation restricts direct extrapolation to human immunity. Accordingly, future studies will incorporate in vitro assays using human monocyte-derived dendritic cells and CD4 ⁺ T cells to assess antigen uptake, processing, and presentation. In addition, evaluation of vaccine efficacy against virulent clinical isolates of VZV remains technically challenging, as VZV exhibits strict human tropism and there is currently no well-established small animal challenge model that reliably supports infection with virulent strains. As a result, direct in vivo protection studies against wild-type VZV are not feasible in conventional mouse models. In this study, the attenuated v-Oka strain was therefore used in neutralization assays as a standardized and widely accepted surrogate to assess functional antibody responses. Furthermore, the current mouse model reflects primary VZV immunization rather than viral reactivation, and thus represents a proof-of-concept for improved immunogenicity rather than clinical equivalence to RZV. Future investigations using humanized or reactivation-prone models will be essential to determine whether these immunological advantages can translate into durable protection against herpes zoster in humans.
The Nano-gEVZV vaccine, built upon the well-characterized p239 scaffold, substantially enhances the performance of subunit vaccines and shows strong potential for application. This advantage arises from the proven success of p239 in the licensed hepatitis E vaccine, which ensures reliable antigen production and a well-established clinical safety profile [39]. Moreover, the incorporation of a nanobody-based binding strategy enables flexible antigen substitution, providing a versatile platform for the development of nanoparticle vaccines against diverse viral pathogens. Despite these advantages, two potential risks warrant consideration: the stability of non-covalently assembled nanoparticles under physiological conditions and the immunogenic safety of the nano-binder [40,41]. With respect to stability, there remains a theoretical concern that nanoparticles could partially dissociate in vivo, thereby reducing their immunogenic effectiveness [42]. Although in vitro assays demonstrated high-affinity interactions between the nano-binder and p239, direct in vivo evidence confirming nanoparticle integrity is still limited. Nevertheless, current data collectively suggest that this risk is minimal. Advances in structural biology and protein engineering now enable detailed characterization of nanobody–particle interfaces [43–45], supporting rational strategies to enhance stability, such as engineering disulfide linkages [46]. Regarding safety, the multivalent display of nanobody binders on the nanoparticle surface raises theoretical concerns about immunogenicity in the context of vaccination [47–50]. However, camelid-derived nanobodies share substantial structural and sequence homology with human immunoglobulin domains, providing a favorable basis for immune tolerance. Established humanization strategies can further reduce residual immunogenicity while preserving binding affinity and specificity. [51–54]. In the Nano-gEVZV design, the nanobody is largely shielded by the densely displayed VZV gE antigen on the particle surface, which is expected to limit immune exposure. In addition, recent advances in artificial intelligence–assisted protein design enable the rational or de novo redesign of binding modules to minimize immunogenic sequence features while retaining high-affinity interactions. These approaches offer a forward-looking strategy to further mitigate immunogenic risk in future iterations of this platform. Taken together, while the above considerations address key theoretical risks associated with nanoparticle stability and nanobody immunogenicity, the present study was not designed to provide a comprehensive safety or toxicological evaluation. Specifically, dedicated in vitro cytotoxicity assays and systematic assessments of animal well-being, were beyond the scope of this proof-of-concept study, which focused primarily on antigen presentation and immunogenicity. Animals were monitored daily for general health and behavior, and no overt adverse effects were observed following immunization. These observations support the general tolerability of the Nano-gEVZV formulation at the tested dose, while highlighting the need for more extensive cellular and systemic safety evaluations in future preclinical development. Collectively, these considerations delineate both the strengths and current limitations of the Nano-gEVZV platform, indicating that while key theoretical risks appear manageable within the present design, further systematic safety and translational studies will be essential to fully define its clinical potential.
The recombinant zoster vaccine Shingrix has established a new benchmark for herpes zoster prevention, demonstrating the power of combining a well-defined antigen—VZV gE—with the potent AS01B adjuvant system. This success underscores a key principle in modern vaccinology: durable protection arises from the synergy between precise antigen design and optimal immune stimulation. Building upon this paradigm, our study introduces a nanoparticle-based formulation of gE (Nano-gEVZV) co-administered with XUA01, an AS01B-like adjuvant. Compared with monomeric VZV gE formulations, Nano-gEVZV elicited stronger humoral and cellular immune responses in mice. Importantly, our results do not suggest that antigen particleization can substitute for adjuvants; rather, they highlight how structural optimization of antigens can act synergistically with adjuvant systems to amplify adaptive immunity. Given that nanoparticle presentation can intrinsically enhance antigen immunogenicity, incorporating adjuvants with milder reactogenicity—such as CF501 or TLR-based adjuvants may help sustain strong cellular and humoral responses while potentially improving tolerability. In this context, CF501—a novel stimulator of interferon genes (STING) agonist—has recently shown the ability to drive potent humoral and cellular immune responses while maintaining a favorable safety profile [55]. The integration of Nano-gEVZV with CF501 or other adjuvants represents a promising direction for future research [56]. Further studies in relevant animal models and clinical trials will be necessary to determine whether this strategy can match the high efficacy and long-term durability of Shingrix while improving vaccine tolerability across diverse populations.
Materials and methods
Ethics statement
The experimental procedures complied with ARRIVE ethical guidelines and received approval from the Xiamen University Animal Ethics Review Committee (approval number XMULAC20240332).
Cell, virus stock, reagents and antibodies
Spodoptera frugiperda (Sf 9) insect cells (ATCC CRL-1711; Thermo Fisher Scientific; Waltham, MA) were cultured in suspension at 28°C using serum-free ESF921 medium (Expression Systems). Human retinal epithelial ARPE-19 cells (ATCC CRL-2302; Manassas, VA,USA) were seeded in 48-well plates and cultivated in a 1:1 mixture of DMEM/F12 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA). Murine dendritic cells (DC2.4, ATCC CRL-10739) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in RPMI-1640 medium. The live-attenuated VZV vaccine (v-Oka strain, Wantai, Beijing, China) was preserved at -80°C and was titrated using a plaque assay prior to experimentation. Recombinant baculovirus was constructed using the BacMagic system (Merck & Co Inc, Kenilworth, NJ, USA) and the progeny virus was stored at 4°C. HEV-p239 protein was produced and provided by Wantai Company (Xiamen, China).
Generation and production of the anti-p239 nanobodies
The immunization of alpacas and the subsequent selection of nanobodies were conducted by Shenzhen Kangti Biotechnology Co., Ltd. The detailed screening process follows the methodology described in the referenced literature [57]. Briefly, this process encompasses four essential steps. Initially, alpacas received three doses of a human hepatitis E vaccine, after which peripheral blood mononuclear cells (PBMCs) were isolated from the blood serum. RNA was then extracted from the PBMCs, converted into cDNA, and inserted into the pComb3XSS phage vector, which was used to transform TG1 bacterial cells, creating a bacterial library. In the third step, helper phages were introduced into this library to facilitate the creation of a phage display library, from which positive clones were identified through a rigorous sequence of selection, amplification, and purification across three rounds. Finally, the sequences of these positive clones were analyzed to identify and select the candidate molecules.
NB-gE plasmids constructions and protein purification
The NB (nanobody, corresponding to the variable domain of a heavy-chain-only antibody, VHH) –gE plasmid encodes the NB fused to the coding sequence of VZV gE. The NB candidates (S1 Table) was designed at the N-terminus of VZV gE (GenBank accession no. NC_001348). The entire sequence was synthesized and subsequently cloned into the baculovirus vector pIEX/Bac-1 by Sangon Biotech (Shanghai, China). The expression and purification of the NB-gE protein followed previously established protocols. Co-transfection of NB-gE plasmids with linearized DNA (deficient in v-cath/chiA genes) was carried out in Sf 9 insect cells, adhering to the manufacturer’s guidelines (Expression Systems, CA, USA). Following four rounds of subculture, the supernatants were harvested. Purification involved centrifuging the supernatant at 7,000 rpm for 15 minutes and subsequent dialysis against phosphate-buffered saline (PBS), pH 7.4, before purification using Ni-Sepharose High Performance 6 resin (GE Healthcare, Boston, MA, USA) and elution with 250 mM imidazole. Protein concentrations in the final purified samples were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). The reactivity of the purified proteins was assessed through indirect enzyme-linked immunosorbent assay (ELISA).
Enzyme-linked immunosorbent assay (ELISA)
gE proteins purified from Sf 9 cells were coated onto the wells of 96-well plates (Corning Inc, Corning, NY, USA) at 100 ng per well and incubated overnight at 4°C. The wells were subsequently blocked with 2% BSA solution at 37°C for 2 hours. Serum samples were serially diluted three-fold starting from a dilution of 1:100 (100 µL) and applied to the wells for 1 hour at 37°C. Following this, the plates were washed and incubated with HRP-labeled secondary antibody diluted 1:5000 for 1 hour (ThermoFisher Scientific, 31430). After three washing steps, 100 µL of peroxidase substrate (o-phenylenediamine dihydrochloride; Sigma-Aldrich) was added to each well, and the reaction was halted after 10 minutes at 37°C by the addition of 2 M sulfuric acid. The absorbance was measured at 450 nm with a reference wavelength of 620 nm using a microplate spectrophotometer (Tecan; Männedorf, Switzerland).
VZV gE protein quantification
The total protein concentrations of Nano-gEVZV and the P28C-gEVZV were quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). In the double-antibody sandwich ELISA, 4A2 served as the capture antibody, while 6H7-HRP was employed as the detection antibody, following the procedure outlined in a previous study [58]. To establish a standard curve, purified gE proteins at known concentrations were utilized (Y = 0.3063*X + 0.07308, R2 = 0.9917). The content of VZV gE on the nanoparticle scaffold was accurately determined using the double-antibody sandwich ELISA technique.
Preparation of XUA01 adjuvant
XUA01 is a liposomal adjuvant system designed to be analogous to AS01B. The formulation was prepared using a microfluidics-based method [59]. In brief, dioleoyl phosphatidylcholine (DOPC, 1 mg), cholesterol (0.25 mg), and 3-O-desacyl-4′-monophosphoryl lipid A (MPLA, 50 μg) (Merck, Darmstadt, Germany) were dissolved in ethanol to generate the organic phase. An aqueous phase was prepared with QS-21 (Quillaja Saponaria, 50 μg) (AVT Pharmaceutical Tech Co., Ltd., Shanghai, China) in phosphate-buffered saline (PBS, pH 6.1). The two phases were combined through microfluidic mixing to promote liposome formation. Following liposome assembly, ethanol was removed by diafiltration against PBS replacement buffer to ensure complete solvent elimination. The resulting liposomal suspension was sterilized by filtration through a 0.22 μm polyethersulfone (PES) membrane. Particle size and polydispersity index (PDI) were characterized by dynamic light scattering (DLS), while liposomal morphology and homogeneity were further examined by transmission electron microscopy (TEM).
Animal welfare measures
To assess antigen distribution and T cell activity, this study employed a standard animal anesthesia device (R510-29, Rayward Life Technologies Inc., Shenzhen, China) for in vivo imaging and ex vivo organ analysis. To minimize signal interference due to animal movement during imaging, mice were anesthetized short-term with isoflurane (Rayward, Shenzhen, China). Anesthesia was administered via the respiratory system at an oxygen flow rate of 0.5 liters per minute, using 3% isoflurane during the induction phase and 1.5% during the maintenance phase. This method is commonly used in murine experiments, as it effectively provides the required depth of anesthesia while reducing stress on the animals. Post-imaging, the animals were humanely euthanized under anesthesia by cervical dislocation, and standard rodent dissection techniques were employed to isolate lymph nodes and spleen tissues for further analysis.
In vivo fluorescence Imaging
To investigate the targeting ability of Nano-gEVZV, 5 μg of Alexa Fluor647-labeled P28C-gEVZV (AF647-gEVZV) or 7.5 μg of Nano-gEVZV loaded with 5 μg of AF647-gEVZV were subcutaneously injected into the hind footpads of mice (n = 3). After isoflurane anesthesia, mice were placed in the left lateral decubitus position, and a small section of hair was removed from the right flank. The lymph nodes were visualized using an IVIS Spectrum system (Caliper, Hopkinton, MA, USA) at predetermined time points. After nine days, the popliteal and inguinal lymph nodes were isolated for ex vivo imaging (n = 3). The fluorescence intensity in the inguinal lymph nodes was quantified using Living Image 4.0 software.
Immunofluorescence assay (IFA)
Immunofluorescence staining was visualized with a Leica TCS SP8 confocal microscope (Leica, Germany), following previously described procedures [60]. DC2.4 cells were seeded into 24-well plates with coverslips at a density of 2 × 10⁵ cells/well. Both Nano-gEVZV and the P28C-gEVZV, labeled with fluorescent dye (Thermo Fisher Scientific), were introduced to the cells at a concentration of 1 μg/mL and incubated at 37°C for one hour. Subsequently, the cells were washed three times with PBS after removal of the cell culture medium. The cell membranes were stained with DiO (Molecular Probes), a green fluorescent membrane dye diluted 1:100, for 30 minutes at 37°C to delineate cellular boundaries, and nuclei were counterstained with DAPI. The fluorescence was observed with a confocal microscope, and images were analyzed using Fiji software (NIH, Bethesda, MD, USA).
Immunization of C57/BL6 mice
C57BL/6 mice maintained under specific pathogen-free (SPF) conditions were randomized into four groups (five mice per group). They were immunized with one of the following formulations: Nano-gEVZV combined with an AS01B-like adjuvant; P28C-gEVZV combined with an AS01B-like adjuvant; the VZV vaccine (Shingrix); or the HEV vaccine (Hecolin). Each vaccine contained 5 μg of VZV gE protein, as verified by sandwich ELISA. The mice received intramuscular injections of 100 μL of the vaccine on days 0 and 28. Blood samples were collected weekly, centrifuged at 13,000 × g for 10 minutes, and the serum was stored at –20 °C until analysis. On day 14 after the final immunization, splenocytes were harvested for enzyme-linked immunospot assay (ELISPOT) and intracellular cytokine staining (ICS) evaluations.
Neutralization assay
VZV-specific neutralizing antibody titers were quantified through a 50% plaque reduction neutralization test (PRNT₅₀), adhering to previously established methodologies with slight modifications [61,62]. Briefly, serum samples were heat-inactivated at 56 °C for 30 minutes. Two-fold serial dilutions of mouse sera (starting at 1:100) were mixed with an equal volume of v-Oka strain VZV (Wantai, Beijing, China) containing approximately 150 plaque-forming units and incubated at 37 °C for one hour to allow neutralization. The serum–virus mixtures were then added to confluent ARPE-19 cell monolayers in 24-well plates and incubated for one hour at 37 °C to facilitate virus adsorption. After adsorption, the inoculum was removed, and cells were overlaid with fresh DMEM/F12 medium containing 1% methylcellulose. Plates were incubated at 37 °C for 72 hours. After incubation, cells were fixed, stained, and plaques were counted. Neutralization titers were defined as the highest serum dilution capable of reducing the number of plaques by 50% compared to the virus control.
Intracellular cytokine staining (ICS) and flow cytometry
VZV gE-specific CD4⁺ and CD8 ⁺ T cells expressing IFN-γ or IL-2 were identified through intracellular cytokine staining (ICS) and flow cytometry, following previously described methods [63]. Single-cell suspensions from spleens (2 × 10⁶ cells) isolated from immunized mice were restimulated in vitro for 6 hours with a 15-mer VZV gE peptide pool featuring an 11-amino-acid overlap (2 μg/mL). During incubation, protein transport inhibitors (BD GolgiPlug; BD Biosciences) were added. After incubation, cells were washed in PBS containing 1% fetal calf serum (FCS) and blocked with Mouse BD Fc Block at 4 °C for 30 minutes. Cells were then stained with PE/Cy7-conjugated anti-mouse CD8 (1:100 dilution; BioLegend, San Diego, CA, USA), FITC-conjugated anti-mouse CD4 (1:100 dilution; BioLegend), and Live/Dead Fixable Aqua stain (Invitrogen, Carlsbad, CA, USA) in a 50 μL mixture. Subsequently, cells were fixed and permeabilized using the Fixation/Permeabilization Solution Kit (BD Biosciences) and stained with APC-conjugated anti-mouse IFN-γ and PE-conjugated anti-mouse IL-2 (1:100 dilution; BioLegend). Following two washes in 1 × Perm Wash solution and resuspension in FCS, cells were analyzed on a BD LSRFortessa X-20 Flow Cytometer (Becton Dickinson). Live cells were selectively gated, with data acquisition focusing on approximately 50,000 events. Analysis was performed using FlowJo software version 10.4.2. Results are presented as the background-corrected mean responses of gE-specific cells, expressed as percentages of the overall frequencies of CD4⁺ or CD8 ⁺ T cells expressing IFN-γ or IL-2.
Enzyme-linked immunospot assay (ELISPOT)
The ELISPOT assay was conducted in accordance with the manufacturer’s instructions (MabTech, Sweden). Specifically, 15-mer peptides of VZV gE were synthesized, ensuring each peptide had an 11-amino-acid overlap to fully represent the gE protein sequence. An optimal concentration of 5 × 10⁵ cells per well was seeded onto precoated ELISPOT plates supplied by MabTech. These cells were exposed to pooled VZV gE peptides and stimulated for 20 hours. Spot formation was initiated as per the manufacturer’s guidelines, with the developed spots scanned and quantified using a CTL-ImmunoSpot S5 reader. The number of spot-forming cells (SFC) was determined by deducting the average number of spots in wells stimulated with PBS. A cocktail of phorbol 12-myristate 13-acetate (PMA, 20 ng/mL) and ionomycin (Sigma-Aldrich, 1 μg/mL) was employed as a positive control, while PBS served as the negative control.
Western blotting
Equal amounts of Sf 9 cell lysates were subjected to SDS-PAGE and electrophoresed for 90 min at 80 V in a Bio-Rad Mini-PROTEAN Tetra system (Bio-Rad Laboratories, Hercules, CA, USA). Gels were then transferred onto nitrocellulose membranes (Whatman, Dassel, Germany) using a Trans-Blot Turbo Transfer System (Bio-Rad Laboratories). The membranes were blocked at room temperature for 1 hour and then incubated with the 1B11 gE-specific antibody. After three washes with PBST (PBS containing 0.1% Tween-20), the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibodies for 30 minutes at room temperature. Following additional washes, protein detection was carried out using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).
Statistical analysis
Statistical analysis and graphing were performed using GraphPad Prism. The statistical details are described in the figure legends. Data were presented as mean ± SD. For comparison between each group with the mean of every other group within a dataset containing more than two groups, one-way ANOVA with Tukey’s test, or Kruskal–Wallis ANOVA with Dunn’s post hoc test were used as appropriate.
Supporting information
S1 Fig. Surface plasmon resonance (SPR) analysis was performed to assess the binding of VHH-gE to HEV-p239 at gradient concentrations.
The binding curves were analyzed using kinetic analysis provided by Biacore Evaluation Software. Data are presented as response units (RU) over time (s). The K_D values were calculated using a 1:1 binding model. Colored lines represent the original curves, while black lines are the fitted curves.
https://doi.org/10.1371/journal.ppat.1013983.s001
(TIF)
S2 Fig. In vivo fluorescence imaging of Nano-gEVZV in mice following subcutaneous footpad inoculation.
Signals from the popliteal and inguinal lymph nodes were analyzed at various time points (n = 3).
https://doi.org/10.1371/journal.ppat.1013983.s002
(TIF)
S3 Fig. Uptake of Nano-gEVZV by dendritic cells and bone marrow–derived cells.
(A) The proportion of glycoprotein E (gE) in Nano-gEVZV was analyzed using SDS-PAGE, providing an accurate measurement of the VZV gE proportion relative to p239 present in Nano-gEVZV. (B) Confocal microscopy images showing enhanced internalization of Alexa Fluor 647–labeled Nano-gEVZV compared with P28C-gEVZV in DC2.4 cells. Cell membranes were stained with DiO (green) and nuclei with DAPI (blue). Scale bar: 20 μm. (C) Flow cytometric analysis of antigen uptake by primary bone marrow–derived cells isolated from C57BL/6 mice after incubation with indicated antigen formulations. (D) Quantitative analysis of the proportion of AF647 ⁺ cells, showing significantly higher uptake of Nano-gEVZV than monomeric gE. Data are presented as mean ± SD. Statistical significance was determined by unpaired two-tailed Student’s t-test; P < 0.05 was considered statistically significant.
https://doi.org/10.1371/journal.ppat.1013983.s003
(TIF)
S4 Fig. Preparation and characterization of adjuvants.
(A) Dynamic light scattering (DLS) analysis of AS01B and XUA01 adjuvants showing comparable hydrodynamic diameters, indicating similar particle size distribution. (B) Macroscopic appearance and representative negative-stain transmission electron microscopy (TEM) images of AS01B adjuvant revealing uniform, spherical vesicles. (C) Macroscopic appearance and representative negative-stain TEM images of XUA01 adjuvant showing morphology consistent with AS01B. Scale bars, 100 nm. Photograph taken by the authors and published under the CC BY 4.0 license.
https://doi.org/10.1371/journal.ppat.1013983.s004
(TIF)
S5 Fig. Neutralizing titers of the antisera were determined using enzyme-linked immunospot (ELISPOT) assays.
Significant differences were observed in the antisera from various groups at different dilutions, with the Nano-gE–induced neutralization titer being the highest.
https://doi.org/10.1371/journal.ppat.1013983.s005
(TIF)
S6 Fig. Characterization of T-cell immune responses using flow cytometry.
(A) Flow cytometry gating strategy to identify different cell populations. (B-C) The proportions of IFN-γ–positive CD4⁺ (B) and CD8⁺ (C) T cells were assessed across various vaccination groups. (D-E) The proportions of IL-2–positive CD4⁺ (D) and CD8⁺ (E) T cells were analyzed among various vaccination groups.
https://doi.org/10.1371/journal.ppat.1013983.s006
(TIF)
S7 Fig. Specific T-cell responses measured by ELISPOT.
(A) IFN-γ–positive spot-forming cells (SFCs) per 100,000 cells. (B) IL-2–positive SFCs per 100,000 cells. Rows represent various vaccination groups, whereas columns correspond to distinct biological individuals. Abbreviations: IFN-γ, interferon-γ; IL-2, interleukin-2; ELISPOT, enzyme-linked immunospot.
https://doi.org/10.1371/journal.ppat.1013983.s007
(TIF)
S1 Table. Amino acid sequences of six alpaca-derived nano-binders (NBs) selected for anchoring VZV gE to the p239 nanoparticle scaffold.
https://doi.org/10.1371/journal.ppat.1013983.s008
(TIF)
Acknowledgments
We thank Prof. Tong Cheng and Prof. Jun Zhang for their financial support of this work. We thank Figdraw for assistance with figure preparation. We thank Dr. D. Wang from Xiamen Innovax Biotech Co., Ltd. for providing the p239 particles.
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