Manganese Mineralization of Pathogenic Viruses as a Universal Vaccine Platform

Figures

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  Figure 1. Physiochemical characterization of MnP‐encapsulated IAV. a) TEM image of IAV@Mn. Samples were negatively stained with phosphotungstic acid. Scale bar = 100 nm. b) EDS analysis and SEM image of IAV@Mn and in situ elemental mapping of Mn, P, and O on the nanoparticle surface. Scale bar = 1 µm. c) Mean diameter of IAV and IAV@Mn measured via DLS. d) Zeta potential of IAV and IAV@Mn measured via NTA. e) FTIR analysis of IAV@Mn; Mn─O─H bond was detected at 634.04 cm−1. f) XRD analysis of IAV@Mn.

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  Figure 2. Attenuation of IAV@Mn in vitro. a) Indirect fluorescence assay of IAV and IAV@Mn in A549 cells (MOI = 0.01) at 48 h.p.i. Cell nuclei were stained by DAPI (blue), and the IAV NP was stained by IAV NP‐specific antibody (green). Scale bar = 100 µm. b) Growth curve of IAV and IAV@Mn in MDCK cells (MOI = 0.01). Virus in supernatant was detected using plaque assay, and the intracellular RNA accumulation was detected by qPCR. c) Indirect fluorescence assay of IAV and IAV@Mn in A549 cells (MOI = 0.01) at 48 h.p.i. d) Growth curve of IAV and IAV@Mn in A549 cells (MOI = 0.1). e) Heat map of RNA‐seq analysis. THP‐1 cells were infected with IAV or IAV@Mn at 0.1 MOI. The heat map was generated by calculating treated RPKM/control RPKM. f) mRNA transcription level analysis of cGAS and STING expression was detected at 6 h.p.i; IFN‐β, ISG15, and ISG54 expression were detected at 24 h.p.i. Cell experiment data are presented as mean ± SD, n = 3, and P‐values were calculated using one‐way or two‐way ANOVA with Bonferroni correction, *P<0.05, **P<0.01, ****P<0.0001.

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  Figure 3. Attenuation of IAV@Mn in vivo. PBS was set as the control, and 5–6‐week‐old female BALB/c mice were intranasally infected with 2 × 104 PFU IAV or IAV@Mn. a) Percent survival of mice (n = 10). Data are presented as mean ± SD, n = 10, and P‐values were calculated using a logrank test, ***P<0.001. b) Replication of IAV and IAV@Mn in the lungs of mice. Tissues were collected at 3 and 6 d.p.i. Data are presented as mean ± SD, n = 3, and P‐values are calculated using two‐way ANOVA with Bonferroni correction, *P<0.05, ****P<0.0001. c) H&E staining of lung pathology (20×). Arrows indicate pathological tissue. Scale bar = 50 µm. d) qPCR analysis of cGAS‐STING pathway in the lung at 24 h.p.i. RNA accumulation was calculated using GAPDH as a reference gene and folded by PBS as 1. Data are presented as mean ± SD, n = 4. e) Serum cytokine levels at 24 h.p.i. The results were detected using Luminex 200 (n = 3).

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  Figure 4. Immunogenicity and immune correlates of protection of IAV@Mn in mice. Three‐week‐old female BALB/c mice were intranasally infected with 104 PFU IAV@Mn. PBS was used as the placebo. a) Schematic diagram of IAV@Mn immunization, sample collection, and immunological assays. b) IAV HA‐specific IgG antibody titers measured by ELISA. Data are presented as mean ± SD, n = 6, and P‐values were calculated using unpaired t‐tests, ***P<0.001. c) Neutralizing antibody titers measured by PRNT. Data are presented as mean ± SD, n = 6, and P‐values were calculated using unpaired t‐tests, ***P<0.001. d) Production of IFN‐γ and IL‐2 in splenocytes measured by ELISPOT. Data are presented as mean ± SD, n = 3, and P‐values were calculated using two‐way ANOVA with Bonferroni correction, ****P<0.0001. e) Production of IFN‐γ, TNF‐α, and IL‐2 in splenocytes measured by flow cytometry. Data are presented as mean ± SD, n = 3, and P‐values were calculated using two‐way ANOVA with Bonferroni correction, **P<0.01, ***P<0.001, ****P<0.0001. f) Percent survival of mice after 105 PFU IAV challenge. Data are presented as mean ± SD, n = 10, and P‐values were calculated using a logrank test, ****P<0.0001. g)…