Proteolysis-targeting influenza vaccine strains induce broad-spectrum immunity and in vivo protection

Shen, Jinying; Li, Jing; Shen, Quan; Hou, Jihuan; Zhang, Chunhe; Bai, Haiqing; Ai, Xiaoni; Su, Yinlei; Wang, Zihao; Zhang, Yunfei; Xu, Beibei; Hao, Jiawei; Wang, Ping; Zhang, Qisi; Ye, Adam Yongxin; Li, Zhen; Feng, Tang; Li, Le; Qi, Fei; Wang, Qikai; Sun, Yacong; Liu, Chengyao; Xi, Xuetong; Yan, Lei; Hong, Hanhui; Chen, Yuting; Xie, Xin; Xie, Jing; Liu, Xiaoheng; Du, Ruikun; Plebani, Roberto; Zhang, Lihe; Zhou, Demin; Church, George; Si, Longlong

doi:10.1038/s41564-024-01908-2

Article
Published: 15 January 2025

Proteolysis-targeting influenza vaccine strains induce broad-spectrum immunity and in vivo protection

Jinying Shen¹^na1,
Jing Li¹^na1,
Quan Shen^1,2^na1,
Jihuan Hou¹^na1,
Chunhe Zhang¹^na1,
Haiqing Bai^3,4^na1,
Xiaoni Ai⁵^na1,
Yinlei Su¹,
Zihao Wang¹,
Yunfei Zhang¹,
Beibei Xu¹,
Jiawei Hao¹,
Ping Wang¹,
Qisi Zhang¹,
Adam Yongxin Ye⁶,
Zhen Li^1,7,
Tang Feng⁸,
Le Li¹,
Fei Qi ORCID: orcid.org/0000-0003-0122-3858¹,
Qikai Wang¹,
Yacong Sun¹,
Chengyao Liu¹,
Xuetong Xi^1,2,
Lei Yan⁹,
Hanhui Hong¹⁰,
Yuting Chen ORCID: orcid.org/0000-0003-0436-8876¹,
Xin Xie^3,4,
Jing Xie ORCID: orcid.org/0000-0001-8156-0322^8,11,
Xiaoheng Liu ORCID: orcid.org/0000-0001-9267-6339¹¹,
Ruikun Du¹²,
Roberto Plebani¹³,
Lihe Zhang⁵,
Demin Zhou ORCID: orcid.org/0000-0003-0339-0959^5,7,
George Church⁶ &
…
Longlong Si ORCID: orcid.org/0000-0002-0504-1659^1,2

Nature Microbiology volume 10, pages 431–447 (2025)Cite this article

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Abstract

Generating effective live vaccines from intact viruses remains challenging owing to considerations of safety and immunogenicity. Approaches that can be applied in a systematic manner are needed. Here we created a library of live attenuated influenza vaccines by using diverse cellular E3 ubiquitin ligases to generate proteolysis-targeting (PROTAR) influenza A viruses. PROTAR viruses were engineered to be attenuated by the ubiquitin–proteasome system, which mediates viral protein degradation in conventional host cells, but allows efficient replication in engineered cell lines for large-scale manufacturing. Depending on the degron–E3 ligase pairs, viruses showed varying degrees of attenuation. In animal models, PROTAR viruses were highly attenuated and elicited robust, broad, strain-dependent humoral, mucosal and cellular immunity. In addition, they provided cross-reactive protection against homologous and heterologous viral challenges. This study provides a systematic approach for developing safe and effective vaccines, with potential applications in designing live attenuated vaccines against other pathogens.

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**Fig. 1: Systematic generation of PROTAR virus vaccines.**

**Fig. 2: Characterization of the viral protein degradation mechanism of PROTAR viruses.**

**Fig. 3: Characterization of PROTAR vaccine in human lung airway on a chip.**

**Fig. 4: Characterization of the in vivo safety of PROTAR vaccines.**

**Fig. 5: Cross-reactive protection against lethal challenge with homologous and heterologous WT viruses in adult mice.**

**Fig. 6: Immune responses and protection efficacy against lethal challenge in old mice, mice with pre-existing immunity and ferrets.**

Generation of a live attenuated influenza A vaccine by proteolysis targeting

Article 04 July 2022

PROTAR Vaccine 2.0 generates influenza vaccines by degrading multiple viral proteins

Article 15 January 2025

Development of broadly protective influenza B vaccines

Article Open access 07 January 2025

Data availability

Change history

12 June 2025
A Correction to this paper has been published: https://doi.org/10.1038/s41564-025-02053-0

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Acknowledgements

This study was supported by the National Key R&D Program of China (grant number 2024YFA0920000 to L.S.), Shenzhen Medical Research Fund (grant number B2303001 to L.S.), the National Natural Science Foundation of China (grant number 82273837 to L.S. and grant number 32101173 to Y.C.), Shenzhen Science and Technology Program (grant number JCYJ20220818101405011 to L.S.), the Strategic Priority Research Program of the Chinese Academy of Sciences (grant number XDB0480000 to C.L.), National Key R&D Program of China (grant number 2022YFC3400203 to Y.C.), China Postdoctoral Science Foundation (grant number 2022M723308 to J.L.), Guang Dong Basic and Applied Basic Research Foundation (grant number 2022A1515110061 to J.L.), and Key Laboratory of Quantitative Synthetic Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences. We are grateful to the Shenzhen Infrastructure for Synthetic Biology for instrument support and technical assistance. We thank F. Yang, X. Shi, H. Li, W. Chen and C. Liu from the Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, and X. Ou from the School of Life Sciences, Southern University of Science and Technology, for helpful suggestions.

Author information

These authors contributed equally: Jinying Shen, Jing Li, Quan Shen, Jihuan Hou, Chunhe Zhang, Haiqing Bai, Xiaoni Ai.

Authors and Affiliations

State key Laboratory of Quantitative Synthetic Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China
Jinying Shen, Jing Li, Quan Shen, Jihuan Hou, Chunhe Zhang, Yinlei Su, Zihao Wang, Yunfei Zhang, Beibei Xu, Jiawei Hao, Ping Wang, Qisi Zhang, Zhen Li, Le Li, Fei Qi, Qikai Wang, Yacong Sun, Chengyao Liu, Xuetong Xi, Yuting Chen & Longlong Si
University of Chinese Academy of Sciences, Beijing, China
Quan Shen, Xuetong Xi & Longlong Si
Xellar Biosystems, Boston, MA, USA
Haiqing Bai & Xin Xie
Henan Academy of Innovations in Medical Science, Zhengzhou, China
Haiqing Bai & Xin Xie
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, China
Xiaoni Ai, Lihe Zhang & Demin Zhou
Department of Genetics, Harvard Medical School, Boston, MA, USA
Adam Yongxin Ye & George Church
Shenzhen Bay Laboratory, Shenzhen, China
Zhen Li & Demin Zhou
West China Hospital, Sichuan University, Chengdu, China
Tang Feng & Jing Xie
Beijing Daxiang Biotech, Beijing, China
Lei Yan
AccuraMed Technology, Guangzhou, China
Hanhui Hong
Institute of Biomedical Engineering, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Chengdu, China
Jing Xie & Xiaoheng Liu
Qingdao Academy of Chinese Medical Sciences, Shandong University of Traditional Chinese Medicine, Qingdao, China
Ruikun Du
Center for Advanced Studies and Technology (CAST), Department of Medical, Oral and Biotechnological Sciences, ‘G. d’Annunzio’ University of Chieti-Pescara, Chieti, Italy
Roberto Plebani

Authors

Jinying Shen
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Jing Li
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Quan Shen
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Jihuan Hou
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Chunhe Zhang
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Yinlei Su
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Ping Wang
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Le Li
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Yacong Sun
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Xuetong Xi
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Yuting Chen
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Xin Xie
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Jing Xie
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Xiaoheng Liu
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Ruikun Du
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Roberto Plebani
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Lihe Zhang
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Demin Zhou
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George Church
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Longlong Si
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Contributions

L.S. conceived this study. L.S., J.S., J.L., Q.S., J. Hou, C.Z., H.B. and X.A. designed the experiments and analysed the data. J.S., J.L., J.H. and C.Z. performed cell culture experiments and collected data with assistance from Q.S., Z.W., J. Hao, P.W., Q.Z., Z.L., L.L., Q.W., Y.Z., Y. Su, C.L., X. Xi and J.X. Q.S. performed animal experiments and collected data with assistance from J.L., J.S., C.Z., P.W. and L.L.; H.B., X.A., B.X., T.F., L.Y., Y. Sun, X. Xie, J.X. and R.P. performed human lung airway-on-a-chip experiments and collected data. J.L., Q.S., A.Y.Y., H.B. and H.H. performed RNA-seq, scRNA-seq, scBCR-seq and data analysis. F.Q., Y.C., J.X., X.L., R.D., R.P., L.Z., D.Z. and G.C. provided relevant materials, technical assistance and insightful input in terms of experiment design, data analysis and writing. L.S. wrote the paper, which all authors edited.

Corresponding author

Correspondence to Longlong Si.

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Competing interests

L.S., J.S., J.L. and Q.S. are inventors on relevant patent applications held by Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences. The other authors declare no competing interests.

Peer review

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Nature Microbiology thanks Alessio Ciulli and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Systematic investigation on the propagation competence of PROTAR viruses in MDCK-TEVp and conventional MDCK.2 cells.

MDCK-TEVp cells or MDCK.2 cells were infected with indicated viruses (MOI = 0.001) for 48 h and supernatants were collected for quantification of viral titers by TCID₅₀ assay. The factors of decrease in viral titers of PROTAR viruses in MDCK.2 cells relative to those in MDCK-TEVp cells are indicated in the graph. The dotted line indicates the detection limit (10² TCID₅₀/mL). Data are shown as means ± s.d. The experiments were independently performed two times.

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Extended Data Fig. 2 Western blots showing co-IP of E3 ubiquitin ligases with the PTD-tagged viral M1 proteins but not the mutated PTD-tagged viral M1 proteins.

HEK293T cells were transfected with constructs expressing Flag- or HA-tagged E3 ubiquitin ligases and either PTD-tagged viral M1 proteins or mutated PTD-tagged viral M1 proteins and collected 36 h post-transfection for co-IP assay (n = 3). WCL, whole-cell lysate.

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Extended Data Fig. 3 Western blots showing ubiquitination of PTD-tagged viral M1 proteins.

HEK293T cells were co-transfected with constructs expressing HA- or Flag-tagged ubiquitin, Flag- or HA-tagged E3 ubiquitin ligases, and PTD-tagged viral M1 proteins or mutated PTD-tagged viral M1 proteins. The cells were treated with 10 μM MG-132 for 6 hours prior to being harvested at 36 h post-transfection. M1 proteins were immunoprecipitated for detection of the levels of ubiquitination (n = 3).

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Extended Data Fig. 4 PTD-mediated proteasomal degradation of viral M1 protein of PROTAR viruses is dependent on their respective E3 ubiquitin ligase.

Conventional cells were transfected with indicated small interfering RNAs (siRNAs) (20 nM) or short hairpin RNAs (shRNAs) (1 µg/mL) for knockdown of each of the E3 ubiquitin ligases or scrambled RNA control (Ctrl RNA), and 24 h later infected with indicated viruses (MOI = 0.1). 48 h after infection, cells were collected for detection of the levels of influenza M1 protein by western blotting (n = 3).

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Extended Data Fig. 5 PTD-mediated proteasomal degradation of viral M1 protein of PROTAR viruses is E3 ubiquitin ligase-dependent.

a, Inhibition of Cullin-RING E3 ubiquitin ligases restored the levels of PTD-tagged viral M1 proteins. HEK293T cells were transfected with constructs expressing indicated proteins for 6 h and cultured in the presence or absence of 1 μM MLN4924 for another 18 hours. Cells were harvested and subjected to detection of M1 proteins by western blotting (n = 3). b, Inhibition of E3 ubiquitin ligases by specific inhibitors restored the levels of PTD-tagged viral M1 proteins. HEK293T cells were transfected with constructs expressing indicated proteins for 48 h and cultured in the presence or absence of indicated inhibitors for another 6 hours. Cells were harvested and subjected to detection of M1 proteins by western blotting (n = 3).

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Extended Data Fig. 6 Humoral immune responses induced by PROTAR vaccines in adult mice.

a-b, Titers of NT (a) and HI (b) antibodies in sera of C57BL/6 J mice at day 21 post-vaccination with 10⁵ TCID₅₀ of indicated vaccines (n = 5 mice per group). Data are shown as means ± s.d.; one-way ANOVA with Dunnett’s multiple comparisons test; black P values indicate the statistical comparison to Vehicle group; orange P values indicate the statistical comparison to IIV group; red P values indicate the statistical comparison to M1-PTD. c-d, Titers of IgG antibodies against viral surface protein HA (c) and internal conserved protein NP (d) in sera of C57BL/6 J mice at day 21 post-vaccination with 10⁵ TCID₅₀ of indicated vaccines (n = 5 mice per group). Data are shown as means ± s.d.; one-way ANOVA with Dunnett’s multiple comparisons test; black P values indicate the statistical comparison to Vehicle group; orange P values indicate the statistical comparison to IIV group; blue P values indicate the statistical comparison to CAIV group; red P values indicate the statistical comparison to M1-PTD.

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Extended Data Fig. 7 Mucosal immune responses induced by PROTAR vaccines in adult mice.

Titers of IgA antibodies against whole influenza virions (a), HA (b), NA (c), and NP (d) in bronchoalveolar lavage (BAL) of C57BL/6 J mice at day 21 post-vaccination with 10⁵ TCID₅₀ of indicated vaccines (n = 5 mice per group). Data are shown as means ± s.d.; one-way ANOVA with Dunnett’s multiple comparisons test; black P values indicate the statistical comparison to Vehicle group; orange P values indicate the statistical comparison to IIV group; blue P values indicate the statistical comparison to CAIV group; red P values indicate the statistical comparison to M1-PTD.

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Extended Data Fig. 8 T cellular immune responses induced by PROTAR vaccines in adult mice.

a, Viral M1 antigen peptide-specific T cell responses in lungs of C57BL/6 J mice at day 7 post-vaccination with 10⁵ TCID₅₀ of indicated vaccines (n = 5 mice per group), measured by enzyme-linked immunospot (ELISpot) assay. M1_128-135 (MGLIYNRM) antigen peptide was used as the stimuli. Data are shown as means ± s.d.; one-way ANOVA with Dunnett’s multiple comparisons test; black P values indicate the statistical comparison to Vehicle group; orange P values indicate the statistical comparison to IIV group; blue P values indicate the statistical comparison to CAIV group; red P values indicate the statistical comparison to M1-PTD. b-c, Viral NP antigen peptide-specific T cell responses in lungs (b) and spleens (c) of C57BL/6 J mice at day 7 post-vaccination with 10⁵ TCID₅₀ of indicated vaccines (n = 5 mice per group), measured by enzyme-linked immunospot (ELISpot) assay. NP_366-374 (ASNENMETME) antigen peptide was used as the stimuli. Data are shown as means ± s.d.; one-way ANOVA with Dunnett’s multiple comparisons test; black P values indicate the statistical comparison to Vehicle group; orange P values indicate the statistical comparison to IIV group; blue P values indicate the statistical comparison to CAIV group; red P values indicate the statistical comparison to M1-PTD.

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Extended Data Fig. 9 Analysis of viral M1 antigen presentation of PROTAR vaccines.

a, Viral M1 antigen presentation of PROTAR vaccines, IIV, CAIV, and WT virus. Raw264.7 cells were infected with indicated vaccines or viruses (MOI = 0.1) for 6 hours and detected for M1 antigen presentation on the surface of Raw264.7 cells by anti-M1 antibody (left) or an anti-M1 peptide (MGLIYNRM) antibody (right) using flow cytometry. Data are shown as means ± s.d.; n = 3 biological replicates; one-way ANOVA with Dunnett’s multiple comparisons test; black P values indicate the statistical comparison to WT group; orange P values indicate the statistical comparison to IIV group; blue P values indicate the statistical comparison to CAIV group; red P values indicate the statistical comparison to M1-PTD. b, Graphs showing the relevance between PTD-mediated antigen presentation and the levels of PROTAR vaccine-induced immune responses, including IgG antibody responses (top left), IgA antibody responses (top middle and right), T cellular responses against M1 antigen in lungs (bottom left), T cellular responses against NP antigen in lungs (bottom middle), and T cellular responses against NP in spleens (bottom right) (n = 5 mice per group). The same data for M1 antigen presentation are used in all the graphs.

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Extended Data Fig. 10 Characterization of B cell responses induced by PROTAR vaccines in adult mice.

a, Graphs showing numbers of HA- or NP-binding GC B cells per 20,000 cells in spleens of C57BL/6 J mice at day 14 post-vaccination with 10⁵ TCID₅₀ of indicated vaccines (n = 5 mice per group). Data are shown as means ± s.d.; one-way ANOVA with Dunnett’s multiple comparisons test. b, Graphs showing numbers of HA- or NP-binding memory B cells per 20,000 cells in spleens of C57BL/6 J mice at day 14 post-vaccination with 10⁵ TCID₅₀ of indicated vaccines (n = 5 mice per group). Data are shown as means ± s.d.; one-way ANOVA with Dunnett’s multiple comparisons test. c, Graphs showing numbers of HA- or NP-binding plasma cells per 20,000 cells in spleens of C57BL/6 J mice at day 14 post-vaccination with 10⁵ TCID₅₀ of indicated vaccines (n = 5 mice per group). Data are shown as means ± s.d.; one-way ANOVA with Dunnett’s multiple comparisons test. d. Graphs showing numbers of influenza antigens (HA or NP)-specific IgG antibody-secreting plasma cells in spleens or lungs of C57BL/6 J mice at day 14 post-vaccination with 10⁵ TCID₅₀ of indicated vaccines (n = 5 mice per group). Data are shown as means ± s.d.; one-way ANOVA with Dunnett’s multiple comparisons test. e, Graphs showing numbers of influenza antigens (HA or NP)-specific memory B cells in spleens or lungs of C57BL/6 J mice at day 14 post-vaccination with 10⁵ TCID₅₀ of indicated vaccines (n = 5 mice per group). Memory B cells from mouse spleens or lungs were stimulated to proliferate and differentiate into antibody-secreting cells by R848 (1 µg/mL) and recombinant mouse (rm)IL-2 (10 ng/mL) in vitro, and levels were subsequently measured by B-cell ELISpot assay. Data are shown as means ± s.d.; one-way ANOVA with Dunnett’s multiple comparisons test. f, Immunofluorescence images showing GC formation in lungs (left) and spleens (right) of C57BL/6 J mice at day 14 post-vaccination with 10⁵ TCID₅₀ PTD^β-TrCP. Representative images are shown; n = 3 biological replicates. Green, GL7; Red, B220; Blue, DAPI-stained nuclei. Scale bar, 100 µm.

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Shen, J., Li, J., Shen, Q. et al. Proteolysis-targeting influenza vaccine strains induce broad-spectrum immunity and in vivo protection. Nat Microbiol 10, 431–447 (2025). https://doi.org/10.1038/s41564-024-01908-2

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Received: 28 March 2023
Accepted: 06 December 2024
Published: 15 January 2025
Issue date: February 2025
DOI: https://doi.org/10.1038/s41564-024-01908-2

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