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Generation of a live attenuated influenza A vaccine by proteolysis targeting

Nature Biotechnology volume 40, pages 1370–1377 (2022)Cite this article

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Abstract

The usefulness of live attenuated virus vaccines has been limited by suboptimal immunogenicity, safety concerns or cumbersome manufacturing processes and techniques. Here we describe the generation of a live attenuated influenza A virus vaccine using proteolysis-targeting chimeric (PROTAC) technology to degrade viral proteins via the endogenous ubiquitin–proteasome system of host cells. We engineered the genome of influenza A viruses in stable cell lines engineered for virus production to introduce a conditionally removable proteasome-targeting domain, generating fully infective PROTAC viruses that were live attenuated by the host protein degradation machinery upon infection. In mouse and ferret models, PROTAC viruses were highly attenuated and able to elicit robust and broad humoral, mucosal and cellular immunity against homologous and heterologous virus challenges. PROTAC-mediated attenuation of viruses may be broadly applicable for generating live attenuated vaccines.

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Fig. 1: Establishment of the PROTAC virus production system.
Fig. 2: Characterization of PROTAC virus M1-PTD.
Fig. 3: Evaluation of the in vivo safety of PROTAC virus M1-PTD in mice and ferrets.
Fig. 4: Evaluation of the immunogenicity of PROTAC virus M1-PTD in mice and ferrets.
Fig. 5: Characterizations of the protective efficacy of M1-PTD in mice and ferrets.

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Data availability

The gene sequences of WSN influenza virus strain used in this study have been deposited in GenBank under accession numbers CY034138.1, CY034139.1, CY034135.1, CY034134.1, X17336.1, HE802059.1, L25818.1 and CY034136.1. The gene sequences of PR8 influenza virus strain used in this study have been deposited in GenBank under accession numbers CY147541.1, CY147540.1, CY147539.1, CY147534.1, CY147537.1, CY147536.1, CY147535.1 and CY147538.1. 3D structures of M1, PB2, PB1, PA, NP and NS1 have been deposited in the Protein Data Bank (PDB) with PDB IDs: 7JM3, 4WSB, 4WSB, 4IUJ, 2IQH and 4OPH, respectively. Data of VHL expression in human tissues are from GTEx Analysis Release V8 (dbGaP accession number phs000424.v8.p2) (https://gtexportal.org/home/gene/VHL) and also available in the Source Data files. Data of VHL expression in mouse tissues are from RNA sequencing data of E-MTAB-2801 in Expression Atlas and also available in the Source Data files. Data of VHL expression in human lung are from the Human Protein Atlas (http://www.proteinatlas.org/ENSG00000134086-VHL/single+cell+type/lung). All data are available in the manuscript and in Supplementary and Source Data files. No restriction on data availability. Source data are provided with this paper.

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Acknowledgements

We acknowledge the Animal Experimental Management Center, Public Technology Service Platform, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, for assistance with animal experiments. We acknowledge YuBiolab Ltd., Sinovac Biotech Ltd., TsingKe Biotech Ltd., Genewiz lnc., Corregene Biotech Ltd. and Servicebio Ltd. for technical assistance with animal experiments, gene sequencing, plasmid construction, establishment of cell lines and immunohistochemistry experiments. We thank C. Liu, X. Zhang, S. Huang, Y. Ma, B. Zhang and relevant staffs from the Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, for support of technical and experimental platforms. We thank L. Zhu from the Lung Cancer Center, West China Hospital of Sichuan University, for providing paraffin-embedded and frozen sections of human lung tissue. We acknowledge the Genotype-Tissue Expression (GTEx) Project and/or Portal supported by the Common Fund of the Office of the Director of the National Institutes of Health and by NCI, NHGRI, NHLBI, NIDA, NIMH and NINDS. This work was supported by the National Natural Science Foundation of China (grant to L.S., grant no. 11932014 to X.L., and grant no. 81802456 to H.T.), the China-U.S. Collaborative Program on Emerging and Re-emerging Infectious Diseases (grant no. E2900901-02 to W.T.), Guangdong Basic and Applied Basic Research Foundation (grant no. 2019A1515110615 to H.L.), Natural Science Foundation Program of Shaanxi Province of China (grant no. 2020JQ-545 to H.T.), the Key R&D Program of Shanxi Province of China (grant to W.T.), China Postdoctoral Science Foundation (grant no. 2019TQ0206 to H.L.), and the Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences (grant to L.S.).

Author information

Author notes

  1. These authors contributed equally: Longlong Si, Quan Shen, Jing Li.

Authors and Affiliations

  1. CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China

    Longlong Si, Quan Shen, Jing Li, Li Chen, Jinying Shen, Xue Xiao, Le Li, Chunhe Zhang, Zhen Li & Ping Wang

  2. Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, MA, USA

    Haiqing Bai

  3. Institute of Biomedical Engineering, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Chengdu, China

    Tang Feng, Chunhe Zhang & Xiaoheng Liu

  4. Department of Genetics, Harvard Medical School, Boston, MA, USA

    Adam Yongxin Ye & Li Li

  5. Rare Disease Research Unit, Pfizer Inc., Cambridge MA, USA

    Crystal Yuri Oh

  6. Jnana Therapeutics, Boston, MA, USA

    Atiq Nurani

  7. Third Institute of Oceanography, State Oceanic Administration, Xiamen, China

    Siwen Niu

  8. Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA

    Chengxin Zhang & Xiaoqiong Wei

  9. Department of Orthopedics, Peking University Third Hospital, Beijing, China

    Wanqiong Yuan

  10. Beijing Key Laboratory of Spinal Disease, Beijing, China

    Wanqiong Yuan

  11. Engineering Research Center of Bone and Joint Precision Medicine, Beijing, China

    Wanqiong Yuan

  12. Department of Clinical Laboratory, Shenzhen Third People’s Hospital, Southern University of Science and Technology, National Clinical Research Center for Infectious Diseases, Shenzhen, China

    Hao Liao

  13. Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China

    Xiaojie Huang & Ning Wang

  14. College of Veterinary Medicine, Shanxi Agricultural University, Jinzhong, China

    Wen-xia Tian

  15. National and Local Joint Engineering Research Center of Biodiagnosis and Biotherapy, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China

    Hongwei Tian

  16. Center on Advanced Studies and Technology (CAST), Department of Medical, Oral and Biotechnological Sciences, ‘G. d’Annunzio’ University of Chieti-Pescara, Chieti, Italy

    Roberto Plebani

Authors
  1. Longlong Si
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  2. Quan Shen
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Contributions

L.S. conceived this study and designed the experiments. L.S., Q.S. and J.L. performed and analyzed experiments, with other authors assisting with experiments and data analysis. L.S. wrote the manuscript, with all authors providing feedback.

Corresponding author

Correspondence to Longlong Si.

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Competing interests

L.S., Q.S., J.L., L.C., J.S. and X.X. are inventors on the relevant patent applications held by the Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences. The remaining authors declare no competing financial interests.

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Extended data

Extended Data Fig. 1 VHL is widely expressed in tissues of human and mouse.

a, Expression of VHL in human tissues. Data are from GTEx Analysis Release V8 (dbGaP accession number phs000424.v8.p2) (https://gtexportal.org/home/gene/VHL). Box plots are shown as median and 25th and 75th percentiles; points are displayed as outliers if they are above or below 1.5 times the interquartile range. TPM, transcripts per million. b, Expression of VHL in mouse tissues based on RNA-seq data of E-MTAB-2801 in Expression Atlas. Data are presented as mean ± SD; n = 2 for heart and 3 for others. c, Expression of VHL in the human lung. Data are from the Human Protein Atlas (http://www.proteinatlas.org/ENSG00000134086-VHL/single+cell+type/lung)53. pTPM, transcripts per million protein coding genes.

Source data

Extended Data Fig. 2 VHL is widely expressed in lungs of mouse and human.

a, Western blotting images showing the expression of VHL in lungs of BALB/c and C57BL/6 J mice. b, Immunohistochemistry images showing the expression of VHL (brown) in lungs of BALB/c mouse, C57BL/6 J mouse, and human (n = 3). c, Immunofluorescence image showing the expression of VHL in primary human lung alveolar epithelium cultured on Transwells. Green, VHL; blue, nuclei. All experiments were repeated at least two times.

Source data

Extended Data Fig. 3 Schematic structure model of the influenza A virion.

Influenza A virions are enveloped spherical structures with diameters of 80–120 nm54. The genome of each influenza A virion contains eight single-stranded negative-sense RNA segments, encoding 10 main viral proteins: hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1), M2, polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), polymerase acidic protein (PA), nucleoprotein (NP), nonstructural protein 1 (NS1), and nuclear export protein (NEP, also known as NS2). HA and NA are two main antigenic determinants of influenza virus, which are on the surface of the viral envelope and mediate viral entry into host cells and release of progeny virions from host cells, respectively. M2 is the third integral membrane protein, which forms ion channels in virion particles and has roles in viral entry, assembly, and budding. M1, the most abundant protein in the virion, lines the internal surface of the viral lipid bilayer and mediates the interactions between the viral membrane and ribonucleoprotein (RNP). The RNP is a complex structure composed of multiple copies of the viral NP, which wraps eight viral RNA genome segments, and a single RNA polymerase, which binds to the termini of each RNA genome segment. The RNA polymerase has three subunits: PB1, PB2, and PA, which are responsible for viral replication and transcription17,54. NS1 plays an important role in evasion of the host innate immune system and facilitates viral replication. NEP mediates the export of the newly synthesized viral RNP from the nucleus into the cytoplasm.

Extended Data Fig. 4 Structure prediction of PTD-tagged influenza viral proteins according to I-TASSER32.

Green, viral protein; blue, TEVcs linker; orange, proteasome-targeting peptides. Top images showing the overall structures of influenza viral proteins containing PTD, bottom images showing the close-up views on PTD domain in each influenza viral protein. The PDB IDs of M1, PB2, PB1, PA, NP, and NS1 are 7JM3, 4WSB, 4WSB, 4IUJ, 2IQH, and 4OPH, respectively.

Extended Data Fig. 5 Verification of the genetic stability of M1-PTD by sequencing after 20 passages in MDCK-TEVp cells.

The gene sequence used to construct the M1-PTD was used as the template.

Extended Data Fig. 6 M1-PTD infection had no effect on HIF-1α, the endogenous substrate of VHL, even at MOI = 1.0.

MDCK.2 cells were infected with M1-PTD (MOI = 0, 0.01, 0.1, or 1.0) and analyzed for HIF-1α protein levels by western blotting at 48 h post-infection. The experiment was repeated two times.

Source data

Extended Data Fig. 7 The dose-dependent immune responses elicited by M1-PTD in WT mice and the immune responses elicited by M1-PTD in BALB/c Nude mice.

a, HI antibody responses elicited by indicated dosages of M1-PTD in the sera of WT BALB/c (left) and BALB/c Nude (right) mice at day 21 post-vaccination (n = 5). Data are plotted for individual mice and overlaid with mean ± SD; left graph, one-way ANOVA with Dunnett’s multiple comparisons test; right graph, unpaired two-tailed t-test; n.s., not significant. b, NT antibody responses elicited by indicated dosages of M1-PTD in the sera of WT BALB/c (left) and BALB/c Nude (right) mice at day 21 post-vaccination (n = 5). Data are plotted for individual mice and overlaid with mean ± SD; left graph, one-way ANOVA with Dunnett’s multiple comparisons test; right graph, unpaired two-tailed t-test; n.s., not significant. c, Anti-HA antibody responses elicited by indicated dosages of M1-PTD in the sera of WT BALB/c (left) and BALB/c Nude (right) mice at day 21 post-vaccination (n = 5). Data are plotted for individual mice and overlaid with mean ± SD; left graph, one-way ANOVA with Dunnett’s multiple comparisons test; right graph, unpaired two-tailed t-test; n.s., not significant. d, Anti-NP antibody responses elicited by indicated dosages of M1-PTD in the sera of WT BALB/c (left) and BALB/c Nude (right) mice at day 21 post-vaccination (n = 5). Data are plotted for individual mice and overlaid with mean ± SD; left graph, one-way ANOVA with Dunnett’s multiple comparisons test; right graph, unpaired two-tailed t-test; n.s., not significant. Note that M1-PTD did not induce immune response in T cell-deficient BALB/c Nude mice, indicating that T cell response may be the driver for the immunity elicited by M1-PTD. e, Viral M1 antigen-specific T cell responses elicited by indicated dosages of M1-PTD in the lungs of WT C57BL/6 J mice at day 7 post-vaccination (n = 5), measured by ELISpot assay. Two peptides, M1128-135 MGLIYNRM (left) and M158-66 GILGFVFTL (right), were used as the stimuli. IFNγ-expressing cells per million cells were shown. Data are plotted for individual mice and overlaid with mean ± SD; one-way ANOVA with Dunnett’s multiple comparisons test.

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Extended Data Fig. 8 T cell responses induced by M1-PTD and M1 antigen presentation mediated by PTD.

a, CD8 and CD4 T cell responses elicited by 105 PFU of M1-PTD, CAIV, and M1-KO vaccines in lungs of C57BL/6 J mice at day 7 post-vaccination, measured by flow cytometry. Data are plotted for individual mice and overlaid with mean ± SD; n = 3 mice for Vehicle in left graph, 4 mice for CAIV in left graph, and 5 mice for all other groups; one-way ANOVA with Tukey’s multiple comparisons test. b, Representative flow cytometry histograms are shown for lung samples from mice vaccinated with 105 PFU of M1-PTD. Purple, stained cells; red, unstained cells. c and d, PTD-mediated M1 degradation increases M1 antigen presentation in Raw264.7 cells. Raw264.7 cells were infected with M1-PTD or M1KO virus (MOI = 0.1). 24 hours after infection, M1 antigen presentation on the surface of Raw264.7 cells was detected by an anti-M1 peptide (M1128-135: MGLIYNRM) antibody (c) or an anti-M1 antibody (d) using flow cytometry. Data are presented as mean ± SD; n = 3 biological replicates; unpaired two-tailed t-test.

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Extended Data Fig. 9 Characterization of the broad applicability of PROTAC virus vaccine strategy.

a, Generation and characterization of PROTAC virus M1-PTDPR8 by incorporating PTD to M1 protein of influenza A/Puerto Rico/8/1934 (H1N1) virus (PR8). Characterization of the efficient propagation of M1-PTDPR8 in MDCK-TEVp cells and not in conventional MDCK.2 cells by CPE measured by CellTiter-Glo assay. Data are presented as mean ± SD; n = 3 biological replicates. b, Multicycle replication kinetics curves of M1-PTDPR8 in MDCK-TEVp and conventional MDCK.2 cells. The detection limit is 70 PFU/mL. Data are presented as mean ± SD; n = 3 biological replicates.

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Extended Data Fig. 10 Replication of M1-PTD in TEVp- or viral M1-expressing HEK293T cells.

Relative copy numbers of NP gene of M1-PTD viruses in viral supernatants after 2 days of propagation in TEVp-expressing or viral M1-expressing HEK293T cells indicate that M1-PTD can efficiently replicate in both types of cells. Data are presented as mean ± SD; n = 3 biological replicates.

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Si, L., Shen, Q., Li, J. et al. Generation of a live attenuated influenza A vaccine by proteolysis targeting. Nat Biotechnol 40, 1370–1377 (2022). https://doi.org/10.1038/s41587-022-01381-4

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