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. 2024 Aug 9;15(1):6802.
doi: 10.1038/s41467-024-51182-3.

Engineered probiotic Escherichia coli elicits immediate and long-term protection against influenza A virus in mice

Ling Huang #  1   2   3 , Wei Tang #  1 , Lina He  1   2 , Mengke Li  1 , Xian Lin  1   2   4 , Ao Hu  1 , Xindi Huang  1   2 , Zhouyu Wu  1   3 , Zhiyong Wu  1   3 , Shiyun Chen  5 , Yangbo Hu  6   7   8
Affiliations

Engineered probiotic Escherichia coli elicits immediate and long-term protection against influenza A virus in mice

Ling Huang et al. Nat Commun. .

Abstract

Influenza virus infection remains a major global health problem and requires a universal vaccine with broad protection against different subtypes as well as a rapid-response vaccine to provide immediate protection in the event of an epidemic outbreak. Here, we show that intranasal administration of probiotic Escherichia coli Nissle 1917 activates innate immunity in the respiratory tract and provides immediate protection against influenza virus infection within 1 day. Based on this vehicle, a recombinant strain is engineered to express and secret five tandem repeats of the extracellular domain of matrix protein 2 from different influenza virus subtypes. Intranasal vaccination with this strain induces durable humoral and mucosal responses in the respiratory tract, and provides broad protection against the lethal challenge of divergent influenza viruses in female BALB/c mice. Our findings highlight a promising delivery platform for developing mucosal vaccines that provide immediate and sustained protection against respiratory pathogens.

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Conflict of interest statement

Ling H., W.T., S.C., and Y.H. have filed patents related to the EcN-5M2e vaccine described in this manuscript. The remaining authors declare no competing interests.

Figures

Fig. 1
Fig. 1. In vivo evaluation of EcN for locoregional cytokine responses after intranasal administration.
a Representative in vivo IVIS images showing mCherry-labeled EcN (EcN-mCherry, 1 × 108 CFU/10 μL) distribution in key organs post-intranasal delivery at specific times. Organs: Lu (lung and trachea section), St (stomach), Si (small intestine), Li (large intestine), H (heart), L (liver), S (spleen), K (kidney). b Quantitative analysis of relative fluorescence intensity of mCherry-labeled EcN in lungs at the indicated time points (n = 3/group). c Colony forming units (CFU) of bacteria in the lung tissue of mice at the indicated time points after intranasal administration of EcN-mCherry (n = 4/group). d Schematic representation of the experimental protocol. PBS represents the control group and EcN represents the group treated with wild-type EcN strain. e The body weight of mice was monitored for 7 days after intranasal administration with EcN (n = 5/group). This experiment was repeated twice independently. f Hemogram indices of the treated mice at the indicated time points (Day1: PBS n = 8, EcN n = 8; Day3: PBS n = 10, EcN n = 10; Day7: PBS n = 7, EcN n = 8; Day14: PBS n = 6, EcN n = 6). g Lung cytokine levels in PBS vs. EcN-treated mice over 1, 3, 7, and 14 days (Day1: PBS n = 3, EcN n = 5; Day3, Day7 and Day14: PBS n = 4, EcN n = 4). Data are presented as mean ± SEM. Statistical significance was analyzed by two-tailed unpaired t-test. Source data are provided as a Source Data file.
Fig. 2
Fig. 2. Intranasal administration of EcN protects against influenza A virus in BALB/c mice.
a Schematic representation of the experimental procedure for infection with A/Puerto Rico/8/1934 (H1N1) after intranasal treatment with EcN or PBS (D1 group: PBS n = 8, EcN n = 8; D3 group: PBS n = 6, EcN n = 6; D7 group: PBS n = 6, EcN n = 8; D14 group: PBS n = 6, EcN n = 10). Weight loss (b) and survival (c) were monitored for 14 days. Data are presented as mean ± SEM. Source data are provided as a Source Data file.
Fig. 3
Fig. 3. Characterization of the vaccine strain EcN-5M2e.
a Phylogenetic tree for M2e peptides of human-infecting influenza A viruses created using iqtree. b Cartoon model of the 5M2e design and the sequences of M2e from different influenza A virus strains. c Immunogenicity analysis of 5M2e protein. Purified 5M2e protein (10 μg, plus aluminum adjuvant) was immunized subcutaneously three times at 2 weeks intervals (primer-boost-boost). The endpoint titer of IgG to different M2e peptides in serum 1 week after each immunization was determined by ELISA (n = 3/group). Data are presented as mean ± SEM. Statistical significance was analyzed by two-tailed unpaired t-test. d Schematic illustration of the structure of the manipulated probiotic EcN-5M2e vaccine. e Western blot analysis of extracellular 5M2e protein by anti-His or M2e-specific monoclonal 14C2 antibodies. The experiment was repeated three times independently with similar results. Source data are provided as a Source Data file.
Fig. 4
Fig. 4. Antigen-specific systemic immune responses and mucosal immune responses of EcN-5M2e-vaccinated mice.
a Schematic illustration of immunization and sampling in the mouse model. Mice were immunized intranasally with EcN-vec, EcN-5M2e, or PBS control. b Endpoint titers of IgG against H1N1-M2e peptide in serum samples collected one week after primer, the 1st or 2nd booster vaccination as determined by ELISA (n = 10/group). c, d Endpoint titers of IgG1 (c) and IgG2a (d) against H1N1-M2e peptide. Sera were collected one week after the 2nd booster vaccination (n = 10/group). e–g Mucosal IgA levels of nasal lavage fluid (NALF) (e) (n  =  5/group), bronchoalveolar lavage fluid (BALF) (f) (n = 5/group) and serum (g) (n = 8/group) samples collected 2 weeks after the 2nd booster vaccination. Absorbance values at 450 nm (Abs450) in the ELISA assay against H1N1-M2e peptide were recorded. Serum samples were diluted 10-fold in this test. h Percentages of M2e-specific CD4+ T cells producing IFN-γ or IL-2 (n = 5/group). i Percentages of M2e-specific CD8+ T cells producing IFN-γ or IL-2 (n = 5/group). Panels (h, i) are representative data of 2 independent tests. j Cross-binding ability of IgG, IgG1, IgG2a against M2e of various influenza viruses in serum samples taken one week after the 2nd booster vaccination (n = 10/group). The endpoint titer of each sample is displayed. k Cross-binding ability of IgA against M2e of various influenza viruses in NALF (n = 5/group), BALF (n = 5/group) and 10-fold diluted serum (n = 8/group). Data are presented as mean ± SEM. Statistical significance was analyzed by one-way ANOVA followed by Tukey’s multiple comparison test (panels b–i) or two-tailed unpaired t-test (panels j, k). Source data are provided as a Source Data file.
Fig. 5
Fig. 5. Protective effect of EcN-5M2e in the mouse model.
a Schematic illustration of immunization, sampling and influenza A virus challenge in the mouse model. The preimmunized BALB/c mice were challenged intranasally with 10 × LD50 H1N1, 10 × LD50 H3N2 or 5 × LD50 H6N6 in 20 μL PBS. b–d Body weight of mice after challenge with a lethal dose of H1N1 (b) (PBS n = 10, EcN-vec n = 10, EcN-5M2e n = 9), H3N2 (c) (PBS n = 10, EcN-vec n = 8, EcN-5M2e n = 10), or H6N6 (d) (n = 6/group). e–g Survival of mice after challenging with lethal dose of H1N1 (e), H3N2 (f) or H6N6 (g). Panels (b–e) were representative data of 2 independent experiments. h Determination of lung virus titers at 3 or 5 dpi with H1N1 (n = 5/group). i Histopathological analysis of lung tissue sections after H1N1 infection at 5 dpi (bar represents 100 µm). Images are representative of five individual mouse lung sections in each group. Black arrows indicate thickening of the alveolar walls; Yellow arrows indicate the necrotic cellular debris; Green arrows indicate inflammatory cell infiltration; Red arrows indicate vascular blockage. j Pathological scores of lung tissue sections in mice with H1N1 infection at 5 dpi (n = 5/group). Data are presented as mean ± SEM. Statistical significance was analyzed by two-tailed unpaired t-test (panels h, j). Source data are provided as a Source Data file.
Fig. 6
Fig. 6. The underlying protective mechanism of the EcN-5M2e vaccine.
a Schematic illustration of immunization, sampling, and influenza A virus challenge in serum transfer assays. Sera from sequentially vaccinated female BALB/c mice were transferred into naïve BALB/c mice, which were then infected with 3 × LD50 of H1N1 virus. b ELISA was performed to measure endpoint titers of M2e-specific IgG in donors and recipients 18 h after transfer (n = 7/group). c Body weight of mice after a lethal dose of challenge with H1N1 (n = 7/group). d Survival of mice after a lethal dose of challenge with H1N1. e Schematic illustration of immunization, sampling and influenza A virus challenge in the mouse model. f, g Mucosal IgA levels of BALF (f) (n = 5/group) and serum (g) (n = 5/group) samples collected from vaccinated mice infected with H1N1 at 3 or 5 dpi. The absorbance at 450 nm (Abs450) in the ELISA assay against HA protein was recorded. h Heatmap display of cytokine levels in BALF after H1N1 infection at 3 dpi (n = 5/group). i, k Assessment of cytokine levels IFN-γ (i), MCP-1 (j), and TNF-α (k) in BALF samples collected at 3 or 5 dpi (n = 5/group). Data are presented as mean ± SEM. Statistical significance was analyzed by one-way ANOVA followed by Tukey’s multiple comparison test (f, g) or two-tailed unpaired t-test (i–k). Source data are provided as a Source Data file.
Fig. 7
Fig. 7. Correlates to immune protection after CD4+ T cell depletion in BALB/c mice.
a Schematic illustration of CD4+ T cell depletion in EcN-5M2e immunization, sampling and influenza A virus challenge in the mouse model. At 2 weeks after the second boost immunization, EcN-5M2e immunized mice were injected intraperitoneally with anti-mouse CD4 monoclonal antibody (anti-CD4) or rat IgG2a isotype control (ck) on day -3, -2, -1 and 2. On day 0, spleens and lungs were harvested and analyzed by flow cytometry to determine the efficiency of CD4+ T cell depletion. b FCM assessment of the frequency of CD4+ T cells. c Quantification of CD4+ T cells in different groups. Each dot represents one mouse (PBS: n = 5; EcN-5M2e: n = 5; EcN-5M2e+anti-CD4: n = 4; EcN-5M2e+ck: n = 3). Statistical significance was analyzed by two-tailed unpaired t-test. d, e Body weight changes (d) and survival rates (e) of EcN-5M2e-immunized mice after challenged with 10 × LD50 H1N1 influenza viruses in CD4+ T cell depletion assay (PBS: n = 6; EcN-5M2e, EcN-5M2e + anti-CD4, EcN-5M2e + ck: n = 8). Data are presented as mean ± SEM. Source data are provided as a Source Data file.
Fig. 8
Fig. 8. Duration and long-term protection of the EcN-5M2e vaccination against influenza A virus infection.
a Schematic illustration of the immunization, sampling, and experimental procedure. b The endpoint titer of IgG against H1N1-M2e peptide was measured in sera collected at time points 1, 2, 3, 4, 5 and 6 months after the 2nd booster vaccination (n = 8/group). c–e IgA at Abs450 against H1N1-M2e peptide was measured in immune sera (c), BALF (d), or NALF (e) collected at time points 1, 3, and 6 months after the 2nd booster vaccination (PBS: n = 3; EcN-5M2e: n = 5). f, g Mice vaccinated with EcN-5M2e or PBS for 6 months were infected with 5 × LD50 of H1N1 virus, body weight (f) and survival (g) were monitored for 14 days (n = 8/group). Data are presented as mean ± SEM. Statistical significance was analyzed by two-tailed unpaired t-test (panels b–e). Source data are provided as a Source Data file.
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References

    1. Le Sage, V., Lowen, A. C. & Lakdawala, S. S. Block the spread: barriers to transmission of influenza viruses. Annu. Rev. Virol.10, 347–370 (2023). 10.1146/annurev-virology-111821-115447 - DOI - PubMed
    1. Gouma, S., Anderson, E. M. & Hensley, S. E. Challenges of making effective influenza vaccines. Annu. Rev. Virol.7, 495–512 (2020). 10.1146/annurev-virology-010320-044746 - DOI - PMC - PubMed
    1. Krammer, F. & Palese, P. Advances in the development of influenza virus vaccines. Nat. Rev. Drug Discov.14, 167–182 (2015). 10.1038/nrd4529 - DOI - PubMed
    1. Feranmi, F. Universal flu vaccine protects against influenza A and B. Lancet Microbe.3, e902 (2022). 10.1016/S2666-5247(22)00293-2 - DOI - PubMed
    1. Lo Giudice, C. Towards a universal flu vaccine. Nat. Mater.22, 407 (2023). 10.1038/s41563-022-01452-6 - DOI - PubMed

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