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Nanobody-based combination vaccine using licensed protein nanoparticles protects animals against respiratory and viral infections

Nature Biomedical Engineering (2025)Cite this article

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Abstract

Combination vaccines promise to simplify immunization schedules and improve coverage, but remain technically challenging owing to antigen compatibility, immunogenic balance and formulation complexity. Here we report a modular strategy that uses a single-component nanobody binder to noncovalently attach diverse antigens to intact particles from the licensed hepatitis E vaccine. To identify a suitable binder, an alpaca was immunized with the vaccine, and nanobodies were screened via phage display. One nanobody, P1-5B, selectively bound recessed, non-immunodominant sites on the particle surface and enabled stable antigen display without disrupting native immunogenicity. Using this binder, we generated three vaccine formulations displaying five to eleven antigens, including variants from SARS-2 coronavirus, influenza virus and respiratory syncytial virus. These multivalent particles exhibited high-affinity assembly, preserved solubility and induced neutralizing titres up to three log units higher than soluble antigens. In mice, hamsters and non-human primates, the candidate vaccines conferred robust protection and showed a favourable safety profile. This approach introduces a scalable, plug-and-display system for rapid development of customizable combination vaccines.

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Fig. 1: p239:NB-5R, -6R and -RHF were homogeneous nanoparticles.
Fig. 2: p239:NB-5R, -6R and -RHF were demonstrated expected antigenicity.
Fig. 3: p239:NB-D614G induced potent antigen presentation and T–B cell coordination in vitro and in vivo.
Fig. 4: p239:NB-5R induced binding and neutralizing antibodies, and maintained Hecolin immunogenicity in mice.
Fig. 5: p239:NB-6R immunization elicited vaccine-matched and -mismatched neutralizing antibodies and protected against Omicron sublineage BA.5 challenge in hamsters.
Fig. 6: p239:NB-RHF elicited SARS-2, Flu, RSV and HEV neutralizing antibodies in mice and NHPs.
Fig. 7: p239:NB-RHF immunization against challenge with influenza virus, RSV or HEV.

Data availability

All data are available in the main text or the Supplementary Information. The coordinate and structure factors for E2s:P1-5B have been deposited in the Protein Data Bank (accession number 9IMX). Source data are provided with this paper.

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Acknowledgements

We thank D. Wang, A. Lin and C. Cai from Xiamen Innovax Biotech Co., Ltd., for providing the p239 particles and the assistance with the ED50 assay on Hecolin and combination vaccines. We thank H. Zhou (beamline BL02U1 at the Shanghai Synchrotron Radiation Facility) for the assistance in X-ray data collection and processing. We thank Z. Chen (Xiamen University) for the assistance in negative-stain electron microscopy experiment. This work was funded by the National Key Research and Development Program of China (grant 2024YFC2310401 to S.L.), the National Natural Science Foundation (grants 82471861 to T.L. and 81991491 to N.X.), the Natural Science Foundation of Fujian Province (grant 2022J02005 to Z.Z.), the Natural Science Foundation of Xiamen (grant 3502Z20227165 to H.Y.), the China Postdoctoral Science Foundation (grants 2023T160381 to T.L. and GZC20231425 to D.Y.) and the Fundamental Research Funds for the Central Universities (grants 20720220004 to S.L. and 20720220006 to N.X.).

Author information

Author notes

  1. These authors contributed equally: Tingting Li, Wenhui Xue, Sibo Zhang, Hong Wang, Miaolin Lan, Limin Zhang, Min Lin, Ming Zhou, Dong Ying.

Authors and Affiliations

  1. State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, School of Public Health, School of Life Sciences, Xiamen University, Xiamen, China

    Tingting Li, Wenhui Xue, Sibo Zhang, Hong Wang, Miaolin Lan, Limin Zhang, Min Lin, Ming Zhou, Dong Ying, Yarong Zeng, Lingyan Cui, Yifan Yin, Huiqing Chen, Jian Ma, Chang Liu, Jijing Chen, Chen Wang, Zihao Yu, Yanling Chen, Yangtao Wu, Hongjing Liu, Hai Li, Yuyun Zhang, Jinjin Li, Zhen Lu, Zihao Chen, Yali Zhang, Lunzhi Yuan, Lizhi Zhou, Qingbing Zheng, Hai Yu, Jun Zhang, Tong Cheng, Junyu Chen, Yixin Chen, Zizheng Zheng, Ying Gu, Ningshao Xia & Shaowei Li

  2. National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Collaborative Innovation Center of Biologic Products, National Innovation Platform for Industry-Education Integration in Vaccine Research, Xiamen University, Xiamen, China

    Tingting Li, Wenhui Xue, Sibo Zhang, Hong Wang, Miaolin Lan, Limin Zhang, Min Lin, Ming Zhou, Dong Ying, Yarong Zeng, Lingyan Cui, Yifan Yin, Huiqing Chen, Jian Ma, Chang Liu, Jijing Chen, Chen Wang, Zihao Yu, Yanling Chen, Yangtao Wu, Hongjing Liu, Hai Li, Yuyun Zhang, Jinjin Li, Zhen Lu, Zihao Chen, Yali Zhang, Lunzhi Yuan, Lizhi Zhou, Qingbing Zheng, Hai Yu, Jun Zhang, Tong Cheng, Junyu Chen, Yixin Chen, Zizheng Zheng, Ying Gu, Ningshao Xia & Shaowei Li

  3. State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong, China

    Yi Guan

  4. Joint Institute of Virology (Shantou University and University of Hong Kong), Guangdong-Hongkong Joint Laboratory of Emerging Infectious Diseases, Shantou University, Shantou, China

    Yi Guan

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  1. Tingting Li
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  2. Wenhui Xue
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  3. Sibo Zhang
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Contributions

Conceptualization: T.L., W.X., S.Z., Y. Gu, N.X. and S.L. Design: T.L., W.X., S.Z. and S.L. Data collection: T.L., W.X., S.Z., H.W., M. Lan, L. Zhang, M. Lin, M.Z., D.Y., Y. Zeng, L.C., Y.Y., H.C., J.M., C.L., Jijing Chen, C.W., Z.Y., Yanling Chen, Y.W., H. Liu, H. Li, Yuyun Zhang, J.L., Z.L. and Z.C. Analysis: T.L., W.X., S.Z., H.W., M.L., L.Z., M.L., M.Z., D.Y., J.C., Z.Z. and S.L. Preparation of the paper: T.L., W.X. and S.L. Discussion and interpretation of the results: T.L., W.X., S.Z., H.W., L. Zhang, M. Lin, M.Z., D.Y., Yali Zhang, L.Y., L. Zhou, Q.Z., H.Y., J.Z., T.C., Junyu Chen, Yixin Chen, Y. Guan, Z.Z., Y. Gu, N.X. and S.L. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the paper.

Corresponding authors

Correspondence to Tingting Li, Junyu Chen, Zizheng Zheng, Ying Gu, Ningshao Xia or Shaowei Li.

Ethics declarations

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 remaining authors declare no competing interests.

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Peer review information

Nature Biomedical Engineering thanks Kim Good-Jacobson and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Comprehensive characterization of the top eight nanobodies fused with the SARS-CoV-2 D614G receptor-binding domain (RBD).

a SDS-PAGE analysis presented molecular weights and purity of the nanobody fusion protein. b Western blot analysis confirmed the integrity of nanobody fusion proteins using an anti-His tag antibody as detection antibody. c Affinity constants of the nanobody fusion proteins against the HEV p239 antigen were measured by fitting association / dissociation SPR response curves. d HPSEC profiles display formation of HEV p239 particles in complex with the eight nanobody fusion proteins, reflecting the yield and homogeneity of these complexes. A red star notation highlighted the favorable generation of p239:P1-5B-RBD without nanobody binding-triggered aggregation or precipitation during complex formation. All experiments were conducted in duplicate, except for the SPR detection, which was performed once. The data from the experiments provide representative results.

Source Data

Extended Data Fig. 2 Structure determination of the immune complex E2s:P1-5B and characterization of p239:NB-D614G and FR-D614G particles.

a Amino acid sequences of p239 and P1-5B. b SDS-PAGE analysis and HPLC profile and of E2s:P1-5B complex. Crystal of the E2s:P1-5B complex used in X-ray diffraction data collection. c Critical interactions between E2s and P1-5B. d Superimpositions of p239 dimer model with the immune complex structures of E2s:8C11 (PDB no: 3KRD), E2s:8G12 (PDB: 4PLK) and E2s:P1-5B, according to the alignment of E2s domain structure. e P1-5B binding sites being distanced from structurally identified HEV immunodominant epitopes such as 8G12 and 8C11. f Schematic showing multiple single-species NB-D614Gs bind to the surface of p239 particle. g SDS-PAGE analysis of p239, unbound NB-D614G, and p239:NB-D614G nanoparticle. h HPSEC analysis of p239:NB-D614G nanoparticle and its base p239 particle. i Negative stain electron microscopy (EM) showing morphology and size of p239:NB-D614G nanoparticle. j Sedimentation coefficient and distribution in solutions of p239:NB-D614G nanoparticle and its base p239 particle were determined by analytical ultracentrifugation (AUC) sedimentation velocity method. k Mutated P1-5B nanobodies fusion protein (muNB-D614G) designed to disrupt binding, the result confirm that the specific interaction is exclusively mediated by the nanobody. l SDS-PAGE analysis of ferritin (FR), unconjugated D614G receptor-binding domain (RBD), and ferritin-D614G RBD nanoparticle (FR-D614G). RBD-conjugated ferritin resolved at the band representing covalent association of RBD and ferritin with their summed molecular weight. m Hydrated diameters of FR-D614G and FR nanoparticles were determined by dynamic light scattering (DLS). n Negative-stain electron microscopy showing morphology and size of FR and FR-D614G nanoparticles. All experiments were conducted in duplicate, except for the X-ray diffraction, which was performed once. The data from the experiments provide representative results.

Source Data

Extended Data Fig. 3 Affinity constants and binding reactivities of nanobody P1-5B and its fusion proteins.

a Affinity constants of P1-5B and its fusion proteins, including NB-receptor-binding domains (NB-RBDs), NB-HA head regions (NB-HAhrs), and NB-prefusion F proteins (NB-preF), against the HEV p239 antigen were measured by fitting association / dissociation SPR response curves. The NB-RBD (D614G) presented here corresponds to the P1-5B-BRD shown in Extended Data Fig. 1c. It is included in the same diagram to facilitate comparison with other proteins. b Antibody reactivities of nanobody P1-5B fusion proteins, NB-RBDs, NB-HAhrs, NB-preF, and their p239-decorating nanoparticles p239:NB-5R, p239:NB-6R, and p239:NB-RHF, against a panel of antibodies or antisera were quantified as half-maximal effective concentration (EC50) or half-maximal effective dose (ED50, Dlution). S309, 6D6, and 7D6, SARS-CoV-2 broad-spectrum neutralizing monoclonal antibody. H4#, human serum targeting H1N1; T62, polycolonal rabbit antibodies against H3N2; 15F3, mouse monoclonal antibody for Influenza B/Victoria lineage; M1#, mouse serum targeting Influenza B/Yamagata lineage. Am22, D25, and Mota, RSV preF-specific human monoclonal antibodies. 8C11, J286 anti-HEV neutralizing monoclonal antibody. All the experiments above were conducted more than once.

Source Data

Extended Data Fig. 4 Gating strategy for flow cytometry analysis.

a Antigen capture by dendritic cells (DC 2.4) in vitro was analyzed using flow cytometry. The gating strategy for positive signals confirmed the successful uptake of both p239:NB-D614G particles and NB-D614G monomers. b Gating strategies for DC and macrophage. Inguinal lymph nodus (LNs) were digested into single cells, the percentages of antigen-specific DCs (CD11c+MHCII+) and macrophages (CD11b+F4/80+) were analyzed. c Gating strategies for GC B cells and Tfh from LNs. The percentages of GC B cells (CD19+CD95+GL7+), Tfh (CD3+CD4+PD-1+CXCR5+) and RBD+ B cells (CD19+B220+DyLight488+) were analyzed. d Gating strategies for memory B cells (IgD−CD38+CD73+CD80+CD19+B220+DyLight488+) from LNs. The percentages of MBCs (CD19+CD95+GL7+) were analyzed. Experiments above were conducted independently twice.

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Extended Data Fig. 5 p239:NB-5R nanoparticles induced immune response against SARS-CoV-2 in mice and hamster.

a p239:NB-5R nanoparticles induced cross-reactive antibodies response in mice. End-point ELISA data for day 56 (14 days after the 2nd boost) for antisera from individual mice showing the maximal dilution titer. b p239:NB-5R nanoparticles induced cross-neutralizing antibodies response in mice. In the box‑and‑whisker plots, the plus sign indicates the median, the top and bottom of the box the interquartile range, and the whiskers the range. Symbols represent individual animals. Dashed lines indicate the assay limit of detection. c End-point ELISA data for prime and boost for antisera showing the maximal dilution titer. (d-e) p239:NB-5R nanoparticles induced cross-neutralizing antibodies response in hamsters for day 28 (d) and 56 (e) (14 days after the 1st and 2nd boost). Antisera from individual hamster showing the geometric mean titers (GMT), presented as geometric mean with standard deviation (SD). H, 10 µg; L, 2 µg dosage. Saline, placebo cohort. The vaccination experiments above were conducted once. The end-point ELISA and neutralization assays were conducted more than twice. Statistical analysis was performed by Kruskal-Wallis test with Dunn’s multiple comparisons.

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Extended Data Fig. 6 Hecolin:NB-D614G and p239:NB-D614G nanoparticles induced immune response against HEV and SARS-CoV-2 in mice, p239:NB-6R induced cross-clade SARS-CoV-2 binding and neutralizing antibodies in hamster.

a Extremely prompt preparation of Hecolin:NB-D614G complex by mixing NB-D614G antigen with aluminum-formulated Hecolin product in vials were carried out to compare the particulate effect for immunogenicity enhancement with that of p239:NB-D614G made otherwise with p239 antigen and then formulated with aluminum adjuvant. End-point ELISA data for day 14 and 28 (14 days after the boost) for antisera from individual mice showing the maximal dilution titer. Neutralization data (ID50) for day 28 (14 days after the 1st boost) for antisera from individual mice. b A control experiment was conducted using a nanobody fusion-antigen mixture prepared with a mutant nanobody fusion protein that lacks the ability to bind to p239. The results effectively rule out any potential adjuvant effects associated with p239 particles. c End-point ELISA and neutralization data for day 56 (14 days after the 2nd boost) for antisera from individual hamster showing the maximal dilution titer and geometric mean titers (GMT). Neutralization titer (ID50) for day 56 (14 days after the 2nd boost) for antisera from individual hamster. Statistical analysis was performed by two-way analysis of variance (ANOVA) tests with Tukey’s multiple comparisons (a, left and middle) or Kruskal-Wallis test with Dunn’s multiple comparisons (a, right; c and d). The vaccination experiments in mice were conducted twice. The vaccination experiment in hamster were conducted once. The end-point ELISA and neutralization assays were conducted more than twice. Data are presented as geometric mean ± SD. Al, aluminum adjuvant; Saline, placebo cohort.

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Extended Data Fig. 7 Toxicology study of p239:NB-RHF vaccine candidate in mice and monkey.

a Body weight changes after administrations of four-in-one p239:NB-RHF vaccine candidate in mice. Immunization schedule: 5 mice per group were immunized with either p239:NB-RHF adjuvanted with aluminum or XUA01 (AS01B like), or NB-RHF (equivalent to 17 µg NB-RHF content) for three times. Naïve, unvaccinated mice. Body weight change of individual mice and average body weight change in each cohort was monitored following seven days after each administration. b Potential innate immune responses after administrations of four-in-one p239-RHF vaccine candidate in mice. Levels of plasma cytokines were measured 6 or 24 h after first administration. The cytokine levels at both time points were below the detection limit. c Histopathological analysis (H&E staining) after administrations of four-in-one p239-RHF vaccine candidate in mice. H&E-stained section images were examined and recorded 48 h after first administration. Scale bar = 100 μm. d Body temperatures of each monkey were monitored after 1- 4 h post each vaccination. e Body weights of each monkey were measured after the administration of the three vaccinations. f End-point ELISA for antisera from individual monkey showing the maximal dilution titer against P1-5B nanobody. The experiments in mice above were conducted twice. The experiment in monkey was conducted once. Statistical analysis was performed by Kruskal-Wallis test with Dunn’s multiple comparisons. Data are presented as mean ± SD (d-e) or geometric mean ± SD (f).

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Extended Data Fig. 8 Pathology assessment in lung tissue after RSV challenge in mice.

Pathology score of individual mice was based on a severity scale ranging from 0 to 4, that is 0 indicated no pathological changes; 4 reflected the utmost pathological severity; 1 to 3, suggested severity in median extents. Statistical analysis was performed by Kruskal-Wallis test with Dunn’s multiple comparisons.

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Extended Data Table 1 Data collection and refinement statistics for E2s:P1-5B complex
Full size table
Extended Data Table 2 The neutralization titers and body weight loss in individual hamsters following the BA.5 virus challenge
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Source data for Figs. 1–7 and Extended Data Figs. 1–8.

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Li, T., Xue, W., Zhang, S. et al. Nanobody-based combination vaccine using licensed protein nanoparticles protects animals against respiratory and viral infections. Nat. Biomed. Eng (2025). https://doi.org/10.1038/s41551-025-01529-y

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