Journal: Human Vaccines & Immunotherapeutics

Article Title: Mucosal immunization with an adenoviral vector expressing a prefusogenic F protein protects the upper and lower respiratory tracts of cotton rats against live respiratory syncytial virus challenge

doi: 10.1080/21645515.2026.2623573

Figure Lengend Snippet: ChAd68-PreF induces strong neutralization antibody titers against RSV A and RSV B subtypes. serum 60% plaque-reduction neutralization titers (PRNT 60 ) against RSV A2 (A), RSV B (B) and RSV Long (C) determined at days 0, 56 and 61. Symbols represent individual cotton rats; bars show mean ± SD. Statistics: data were log-transformed and analyzed by two-way repeated measures ANOVA with Tukey’s multiple-comparisons test, considering treatment group and time as factors (* p < .05; *** p < .001; **** p < .0001).

Article Snippet: Neutralization assays in cotton rats were performed using the following strains: RSV A2 (ATCC VR-1540), RSV B (strain 18537; ATCC VR-1580PQ), and RSV Long (ATCC VR-26).

Techniques: Neutralization, Transformation Assay

Journal: Microbiology Spectrum

Article Title: Cathepsin S contributes to influenza-induced lung injury by driving inflammation, promoting apoptosis, and disrupting epithelial barrier integrity

doi: 10.1128/spectrum.01128-25

Figure Lengend Snippet: Pathogenesis dynamics and transcriptomic changes during influenza infection in mice. ( A ) Left: the PCA for the sequencing samples at 5 dpi with various infection dose. Right: the PCA for the sequencing samples with 100 or 1,000 PFU PR8 IAVs infection during the time post-infection (1, 3, 5, and 7 dpi). ( B ) Volcano plots of the DEGs between PBS and IAVs treated groups (10 1 , 10 2 , 10 3 , and 10 4 PFU, respectively). The abscissa represents the log 2 FC, the ordinate represents the −log 10 ( P adj). Red points are the upregulated DEGs defined by the absolute value of FC > 2 and P adj < 0.05, while blue points are the downregulated DEGs. ( C and D ) Heatmap clustering of key extracellular-matrix-related genes from overlap DEGs across various infection doses at 5 dpi ( C ) and across multiple timepoints in the 100 PFU infection group ( D ). Expression levels are shown as log 2 -transformed FPKM values after centralization correction, highlighting the top 30 genes with significant changes relative to the PBS group. ( E ) WGCNA module identification and trait correlation, positive and negative correlations are indicated by red and blue colors, respectively. ( F ) Gene interaction network within the ME-darkred module, where node color depth indicates the number of connections. The central area highlights candidate hub genes.

Article Snippet: The Influenza A/Puerto Rico/8/1934 (H1N1) (PR8) virus (ATCC, VR-95PQ) was propagated in 9–11-day-old embryonated chicken eggs and titrated in MDCK cells using either plaque assays or 50% tissue culture infective dose (TCID 50 ) assays ( , ).

Techniques: Infection, Sequencing, Expressing, Transformation Assay

Journal: Microbiology Spectrum

Article Title: Cathepsin S contributes to influenza-induced lung injury by driving inflammation, promoting apoptosis, and disrupting epithelial barrier integrity

doi: 10.1128/spectrum.01128-25

Figure Lengend Snippet: CTSS is upregulated by influenza infection in mouse lung. ( A ) CTSS mRNA expression in lung tissues from mice ( n = 3 per group) inoculated with either PBS or 100 PFU of PR8 virus, quantified by RT-qPCR. ( B ) Western blotting analysis of proteins in lung tissues from mice inoculated with PBS or 100 PFU of PR8 virus. ( C ) CTSS activity in BALF and lung homogenates from mice inoculated with PBS or 100 PFU of PR8, measured using a CTSS Activity Assay Kit. ( D ) Left panel: comparison of CTSS mRNA expression levels in the lungs of mice infected with 5 PFU and 100 PFU of IAV, determined by RT-qPCR. Right panel: western blotting analysis of activated CTSS protein levels in lung tissues from mice infected with 5 PFU and 100 PFU of PR8. ( E ) Temporal profile of CTSS mRNA expression across various days post-infection, measured via RT-qPCR (left), and western blotting analysis of activated CTSS protein levels across different timepoints post-infection (right). Data are represented as mean ± standard deviation (SD). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: The Influenza A/Puerto Rico/8/1934 (H1N1) (PR8) virus (ATCC, VR-95PQ) was propagated in 9–11-day-old embryonated chicken eggs and titrated in MDCK cells using either plaque assays or 50% tissue culture infective dose (TCID 50 ) assays ( , ).

Techniques: Infection, Expressing, Virus, Quantitative RT-PCR, Western Blot, Activity Assay, Comparison, Standard Deviation

Journal: Microbiology Spectrum

Article Title: Cathepsin S contributes to influenza-induced lung injury by driving inflammation, promoting apoptosis, and disrupting epithelial barrier integrity

doi: 10.1128/spectrum.01128-25

Figure Lengend Snippet: The effects of CTSS inhibitor LY3000328 on influenza infection in mice. ( A ) Schematic diagram of the experimental design. C57BL/6J mice ( n = 5 per group) received intraperitoneal injection of 30 mg/kg LY3000328 or a placebo vehicle twice daily for 5 days. Four hours after the initial dose, mice were intranasally inoculated with 100 PFU of PR8 virus. A mock control group received the vehicle orally twice daily and PBS intranasally. Body weight was monitored daily. Mouse lungs were collected at 5 dpi for the analyses of viral titers, histopathology, and gene expression. ( B ) CTSS activity in lung homogenate among different groups. ( C ) Viral titers in lung homogenates determined using the TCID 50 assay in MDCK cells. ( D ) Body weight change of mice across the experimental period. ( E ) Survival curve of mice across the experimental period. ( F ) Histopathological examination of mouse lungs performed by H&E staining. ( G ) Analysis of mRNA expression levels of cytokines in lung tissues by RT-qPCR. Data are represented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, non-significant.

Article Snippet: The Influenza A/Puerto Rico/8/1934 (H1N1) (PR8) virus (ATCC, VR-95PQ) was propagated in 9–11-day-old embryonated chicken eggs and titrated in MDCK cells using either plaque assays or 50% tissue culture infective dose (TCID 50 ) assays ( , ).

Techniques: Infection, Injection, Virus, Control, Histopathology, Gene Expression, Activity Assay, Staining, Expressing, Quantitative RT-PCR

Journal: Microbiology Spectrum

Article Title: Cathepsin S contributes to influenza-induced lung injury by driving inflammation, promoting apoptosis, and disrupting epithelial barrier integrity

doi: 10.1128/spectrum.01128-25

Figure Lengend Snippet: Influenza infection activates CTSS expression in lung epithelial cells (A549). ( A ) CTSS mRNA expression in A549 cells following infection with PR8 virus at MOIs of 0, 0.1, 0.5, and 1. ( B ) Protein levels of pro-CTSS and activated CTSS in A549 cells 24 hpi with serial MOIs of PR8 virus, assessed by western blotting. ( C ) CTSS activity in the supernatant and cell lysate of A549 cells infected with indicated MOI of PR8 virus at 24 hpi, measured using a CTSS Activity Assay Kit. Data are presented as mean ± SD from three independent experiments. ***, P < 0.001; ****, P < 0.0001; ns, non-significant.

Article Snippet: The Influenza A/Puerto Rico/8/1934 (H1N1) (PR8) virus (ATCC, VR-95PQ) was propagated in 9–11-day-old embryonated chicken eggs and titrated in MDCK cells using either plaque assays or 50% tissue culture infective dose (TCID 50 ) assays ( , ).

Techniques: Infection, Expressing, Virus, Western Blot, Activity Assay

Journal: Microbiology Spectrum

Article Title: Cathepsin S contributes to influenza-induced lung injury by driving inflammation, promoting apoptosis, and disrupting epithelial barrier integrity

doi: 10.1128/spectrum.01128-25

Figure Lengend Snippet: CTSS does not affect PR8 virus replication but reduces cytokine expression in A549 cells. A549 cells were transfected with two different CTSS-siRNAs (siCTSS #1 and siCTSS #2) or a scramble siRNA (siSCR) and then infected with 0.25 MOI of PR8 virus. ( A ) Left panel: representative western Blot analysis of CTSS and viral proteins (HA and NP) expression at 24 hpi, Right panel: intensity scanning of the bands from the left, quantified with data from three independent repeats. ( B ) Viral M gene expression at 24 hpi was quantified by RT-qPCR. ( C ) Viral titers at specified timepoints were measured using the TCID 50 assay. ( D ) The changes in mRNA expression levels of CTSS, TNF-α, RANTES, and IP-10 at 24 hpi were assessed by RT-qPCR. Data are presented as mean ± SD. Two to three independent experimental repeats were conducted for each assay. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, non-significant.

Article Snippet: The Influenza A/Puerto Rico/8/1934 (H1N1) (PR8) virus (ATCC, VR-95PQ) was propagated in 9–11-day-old embryonated chicken eggs and titrated in MDCK cells using either plaque assays or 50% tissue culture infective dose (TCID 50 ) assays ( , ).

Techniques: Virus, Expressing, Transfection, Infection, Western Blot, Gene Expression, Quantitative RT-PCR

Journal: Microbiology Spectrum

Article Title: Cathepsin S contributes to influenza-induced lung injury by driving inflammation, promoting apoptosis, and disrupting epithelial barrier integrity

doi: 10.1128/spectrum.01128-25

Figure Lengend Snippet: CTSS is released into the cytoplasm during influenza infection, thereby regulating apoptosis and epithelial barrier integrity. ( A ) Cell death was assessed by measuring LDH release from non-targeting and CTSS-targeting siRNA transfected A549 cells following PR8 infection at 24 hp i, using an LDH Cytotoxicity Assay Kit (Roche, USA, 91963) according to the manufacturer’s instructions. ( B ) TUNEL staining and quantification of positive cells in non-targeting and CTSS-targeting siRNA-transfected A549 cells after PR8 virus infection at 24 hpi. Representative images (left panel) and statistical data (right panel) are shown. ( C ) Protein levels of CTSS, LAMP1, NP, and HA and PARP1 in the cytoplasm, lysosome, and supernatant of A549 cells infected with PR8 (MOI = 0.1) at 0, 6, 12, and 24 hpi were determined by western blotting. ( D ) CTSS activity in the cytoplasm, lysosome, and supernatant of A549 cells infected with PR8 virus was measured using a CTSS Activity Assay Kit. ( E ) Expression of CTSS, intercellular tight junctions, and cell death markers in non-targeting and CTSS-targeting siRNA transfected A549 cells following PR8 virus infection at 24 hpi was determined by western blotting. Data are represented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: The Influenza A/Puerto Rico/8/1934 (H1N1) (PR8) virus (ATCC, VR-95PQ) was propagated in 9–11-day-old embryonated chicken eggs and titrated in MDCK cells using either plaque assays or 50% tissue culture infective dose (TCID 50 ) assays ( , ).

Techniques: Infection, Transfection, LDH Cytotoxicity Assay, TUNEL Assay, Staining, Virus, Western Blot, Activity Assay, Expressing

Journal: Advanced Science

Article Title: Genome‐Wide Codon Reprogramming Enables a Multifactorially Attenuated Influenza Vaccine with Broad Cross‐Protection

doi: 10.1002/advs.202516448

Figure Lengend Snippet: Construction and characterization of the codon‐reprogrammed influenza virus PR8 rp . A) Schematic of PR8 rp genome design. The virus contains wild‐type PB1, PA, and M segments from PR8, and extensively reprogrammed PB2, HA, NP, NA, and NS segments (PB2 rp , HA rp , NP rp , NA rp , NS rp ) using influenza virus's least‐preferred synonymous codons, retaining original 5′ and 3′ packaging signals (PS). B) Plaque morphology of PR8 and PR8 rp viruses in MDCK cells. C–E) Multi‐step growth curves of PR8 (red circles) and PR8 rp (blue squares) in MDCK cells (MOI = 0.01) (C), embryonated chicken eggs (100 plaque‐forming units (PFU) egg −1 ) (D), and A549 cells (multiplicity of infection (MOI) = 0.01) (E). The dashed line represents the detection limit. Each experiment was performed with three independent biological replicates. F,G) Body weight loss (F) and survival rates (G) of BALB/c mice ( n = 5) after intranasal inoculation with the indicated doses of PR8 or PBS as a negative control (NC). H) LD 50 values of PR8 and PR8 rp calculated by the Reed–Muench method based on data from (F,G) and (I,J). I,J) Body weight loss (I) and survival rates (J) of BALB/c mice ( n = 5) after intranasal inoculation with the indicated doses of PR8 rp or PBS (NC). K) Lung viral titers in mice ( n = 3) 3 days post‐infection (dpi) with PR8 (red circles) or PR8 rp (blue squares) at indicated doses. Viral titers were determined by plaque assays on MDCK cells. Data represent mean ± SEM. For comparisons between two groups with equal variances, unpaired two‐tailed Student's t ‐tests were used. For analysis of growth curves, two‐way ANOVA with Šidák's multiple‐comparison correction was used to compare group means at each time point. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: The A/PR/8/34 (H1N1) (PR8) (VR‐95PQ, ATCC) and A/Aichi/2/68 (H3N2) (VR‐547, ATCC) were maintained in the laboratory.

Techniques: Virus, Infection, Negative Control, Endpoint Dilution Assay, Two Tailed Test, Comparison

Journal: Advanced Science

Article Title: Genome‐Wide Codon Reprogramming Enables a Multifactorially Attenuated Influenza Vaccine with Broad Cross‐Protection

doi: 10.1002/advs.202516448

Figure Lengend Snippet: Protective efficacy of PR8 rp vaccination against homologous PR8 challenge in BALB/c mice. A) Experimental design. BALB/c mice were intranasally inoculated with varying doses of PR8, PR8 rp , or an equal volume of PBS as an unvaccinated negative control (NC). Thirty days later, mice were challenged with 1000 × LD 50 of homologous PR8. Body weight and survival were monitored for 14 days; lungs were collected on days 3 and 5 post‐challenge for viral titration and histopathology. B,C) Body weight changes (B) and survival rates (C) of vaccinated mice ( n = 5 per group) following PR8 challenge. D) Lung viral titers at 3 and 5 dpc, determined by plaque assay on MDCK cells ( n = 3 per group). E) Representative H&E staining of lung tissues collected at 5 dpc from normal mice, PBS controls (NC), and mice vaccinated with indicated doses of PR8 or PR8 rp . Scale bars, 25 µm. F,G) Vaccine safety and effective dose ranges (shaded) for PR8 (F) and PR8 rp (G), showing doses that were non‐lethal upon inoculation and provided complete protection against lethal PR8 challenge. Data are shown as mean ± SEM. p ‐values were calculated using one‐way analysis of variance (ANOVA) followed by Tukey's multiple‐comparison post hoc test. **** p < 0.0001.

Article Snippet: The A/PR/8/34 (H1N1) (PR8) (VR‐95PQ, ATCC) and A/Aichi/2/68 (H3N2) (VR‐547, ATCC) were maintained in the laboratory.

Techniques: Negative Control, Titration, Histopathology, Plaque Assay, Staining, Comparison

Journal: Advanced Science

Article Title: Genome‐Wide Codon Reprogramming Enables a Multifactorially Attenuated Influenza Vaccine with Broad Cross‐Protection

doi: 10.1002/advs.202516448

Figure Lengend Snippet: Protective efficacy of PR8 rp vaccination against heterologous challenges in BALB/c mice. BALB/c mice were intranasally inoculated with PR8 rp at the indicated doses or with PBS as a negative control (NC) and challenged 30 days later with heterologous influenza viruses. A–D) Challenge with A/California/04/2009 (H1N1pdm) at 10 × LD 50 : body weight changes (A, n = 5 per group), survival rates (B, n = 5 per group), lung viral loads at 3 and 5 dpc (C, n = 3 per group), and representative hematoxylin and eosin (H&E)‐stained lung sections (D). E–H) Challenge with A/Aichi/2/1968 (H3N2) at 3 × LD 50 : body weight changes (E, n = 5 per group), survival rates (F, n = 5 per group), lung viral loads at 3 and 5 dpc (G, n = 3 per group), and representative H&E‐stained lung sections (H). Viral titers in lung homogenates were determined by TCID 50 assay on MDCK cells. Dashed lines in C and G indicate the limit of detection. Error bars represent SEM. For lung viral titers, one‐way ANOVA followed by Tukey's multiple‐comparison post hoc test was applied. For analysis of body weight changes over time, two‐way repeated‐measures ANOVA with Tukey's multiple‐comparison post hoc test was performed to evaluate the main effects among groups. Survival curves were analyzed using the log‐rank (Mantel–Cox) test. In the analyses of body weight loss and survival rates, black asterisks denote statistical significance compared with the NC group, while light blue asterisks denote significance compared with the PR8 rp 10 1 PFU group. *, p < 0.05; **, p < 0.01; ns, not significant. Scale bars, 50 µm.

Article Snippet: The A/PR/8/34 (H1N1) (PR8) (VR‐95PQ, ATCC) and A/Aichi/2/68 (H3N2) (VR‐547, ATCC) were maintained in the laboratory.

Techniques: Negative Control, Staining, Comparison

Journal: Advanced Science

Article Title: Genome‐Wide Codon Reprogramming Enables a Multifactorially Attenuated Influenza Vaccine with Broad Cross‐Protection

doi: 10.1002/advs.202516448

Figure Lengend Snippet: PR8 rp ‐induced robust humoral immunity contributes to homologous and heterologous protection. BALB/c mice were intranasally inoculated with various doses of PR8 (A,C) or PR8 rp (B,D) viruses, or PBS as an unvaccinated control (NC). Four weeks post‐vaccination, sera were collected and analyzed by hemagglutination inhibition (HI) (A,B) and microneutralization (MN) (C,D) assays against the homologous PR8 virus and heterologous A/California/04/2009 (H1N1pdm) and A/Aichi/2/1968 (H3N2) viruses. Data are shown as geometric means ± 95% CI ( n = 5), with groups not sharing the same lowercase letter differing significantly ( p < 0.05). For passive transfer experiments, pooled sera from mice vaccinated with PR8 rp (10 1 or 10 4 PFU) or from the NC group were administered intraperitoneally (200 µL) to naïve recipients 2 h before intranasal challenge with either 10 × LD 50 H1N1pdm (E–G) or 3 × LD 50 H3N2 (H–J). Body weight (E,H; n = 5 per group), survival rates (F,I; n = 5 per group), and lung viral titers at 3 days post‐challenge (G,J; n = 3 per group) were determined. Data are shown as mean ± SEM. For lung viral titers, one‐way ANOVA followed by Tukey's multiple‐comparison post hoc test was applied. For analysis of body weight changes, two‐way repeated‐measures ANOVA with Tukey's multiple‐comparison post hoc test was performed to evaluate the main effects among groups. Survival curves were analyzed using the log‐rank (Mantel–Cox) test. In the analyses of body weight loss and survival rates, black asterisks denote statistical significance compared with the NC group, while light blue asterisks denote significance compared with the PR8 rp 10 1 PFU group. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

Article Snippet: The A/PR/8/34 (H1N1) (PR8) (VR‐95PQ, ATCC) and A/Aichi/2/68 (H3N2) (VR‐547, ATCC) were maintained in the laboratory.

Techniques: Control, HI Assay, Virus, Comparison

Journal: Advanced Science

Article Title: Genome‐Wide Codon Reprogramming Enables a Multifactorially Attenuated Influenza Vaccine with Broad Cross‐Protection

doi: 10.1002/advs.202516448

Figure Lengend Snippet: PR8 rp elicits functional homologous and heterologous cellular immune responses in mice. BALB/c mice ( n = 3) were intranasally inoculated with a low (10 PFU) or high (10 4 PFU) dose of PR8 rp , or with PBS as a no‐vaccine control (NC). Four weeks later, splenocytes were harvested and stimulated ex vivo with inactivated PR8, A/California/04/2009 (H1N1pdm), A/Aichi/2/1968 (H3N2), or an NP peptide pool. Intracellular cytokine staining was performed to quantify antigen‐specific CD4⁺ and CD8⁺ T cell responses. A) Gating strategy for identifying cytokine‐secreting CD4⁺ and CD8⁺ T cells. B,C) Frequencies of CD4⁺ T cells producing IFN‐γ (B) or IL‐4 (C). D,E) Frequencies of CD8⁺ T cells producing IFN‐γ (D) or IL‐4 (E). Data represent mean ± SEM. p ‐values were calculated using one‐way analysis of variance (ANOVA) followed by Tukey's multiple‐comparison post hoc test. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: The A/PR/8/34 (H1N1) (PR8) (VR‐95PQ, ATCC) and A/Aichi/2/68 (H3N2) (VR‐547, ATCC) were maintained in the laboratory.

Techniques: Functional Assay, Control, Ex Vivo, Staining, Comparison

Journal: Advanced Science

Article Title: Genome‐Wide Codon Reprogramming Enables a Multifactorially Attenuated Influenza Vaccine with Broad Cross‐Protection

doi: 10.1002/advs.202516448

Figure Lengend Snippet: Genome packaging defects, but not RNA or protein synthesis changes, underlie PR8 rp attenuation A) Translation efficiency of wild‐type (PR8) and reprogrammed (PR8 rp ) PB2, HA, NP, NA, and NS segments outside infection. HEK293T cells were transfected with bicistronic plasmids expressing each segment together with EGFP; protein expression was analyzed by Western blot at 24 h post‐transfection. Ratios indicate protein levels normalized to EGFP and expressed relative to PR8. B) Segment‐specific vRNA copy numbers in purified virions (10 5 PFU), determined by qRT‐PCR. Data are presented as mean ± SD. C) Western blot analysis of structural proteins in purified virions; ratios indicate levels relative to PR8. D) Segment‐specific mRNA and vRNA levels in MDCK cells infected at MOI = 3 for 6 h, quantified by qRT‐PCR and expressed relative to PR8. E) Viral protein expression in infected cell lysates (MOI = 3) at 6 hpi; β‐actin served as loading control. F) Genome coverage profiles from high‐throughput sequencing of infected MDCK cells at MOI = 3 for 6 h. Data are shown as mean read counts from three independent biological replicates. p ‐values were calculated using unpaired two‐tailed Student's t‐ tests. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: The A/PR/8/34 (H1N1) (PR8) (VR‐95PQ, ATCC) and A/Aichi/2/68 (H3N2) (VR‐547, ATCC) were maintained in the laboratory.

Techniques: Infection, Transfection, Expressing, Western Blot, Purification, Quantitative RT-PCR, Control, Next-Generation Sequencing, Two Tailed Test

Journal: Advanced Science

Article Title: Genome‐Wide Codon Reprogramming Enables a Multifactorially Attenuated Influenza Vaccine with Broad Cross‐Protection

doi: 10.1002/advs.202516448

Figure Lengend Snippet: Transcriptomic profiling reveals enhanced activation of host antiviral responses by PR8 rp . A549 cells were infected with PR8 or PR8 rp at an MOI of 3 for 12 h, with mock‐infected cells serving as controls. A) Venn diagram showing the overlap of expressed genes among mock‐, PR8‐, and PR8 rp ‐infected cells. B) Volcano plot of differentially expressed genes between PR8 rp ‐ and PR8‐infected cells (padj < 0.05, |log 2 fold change| ≥ 2). Red and blue dots represent significantly upregulated and downregulated genes, respectively; green dots indicate genes with no significant change. C) Bubble plot of KEGG pathway enrichment analysis of genes upregulated in PR8 rp relative to PR8 infection. Dot color represents adjusted p ‐value, and dot size indicates gene count per pathway. D) Heatmap of the top 50 features for the PR8 rp group in differentially expressed genes based on Gene Set Enrichment Analysis.

Article Snippet: The A/PR/8/34 (H1N1) (PR8) (VR‐95PQ, ATCC) and A/Aichi/2/68 (H3N2) (VR‐547, ATCC) were maintained in the laboratory.

Techniques: Activation Assay, Infection

Journal: Advanced Science

Article Title: Genome‐Wide Codon Reprogramming Enables a Multifactorially Attenuated Influenza Vaccine with Broad Cross‐Protection

doi: 10.1002/advs.202516448

Figure Lengend Snippet: ZAP restricts PR8 rp replication by targeting CpG‐enriched viral RNA and enhancing antiviral gene expression. A) Replication of PR8 and PR8 rp in CRISPR‐Cas9–generated ZAP‐knockout (ZAP‐KO) or control knockout (Ctrl‐KO) A549 cells. Cells were infected at an MOI of 0.01, and supernatant virus titers at 24 hpi were quantified by plaque assay. Data are shown as mean ± SEM. B) Heatmap of relative mRNA levels for viral genes (top) and antiviral host genes (bottom) in PR8 rp ‐ or PR8‐infected ZAP‐KO and Ctrl‐KO cells, normalized to GAPDH and expressed relative to PR8‐infected Ctrl‐KO cells. Values represent means of three biological replicates. p ‐values were calculated using unpaired two‐tailed Student's t ‐tests. Asterisks indicate statistically significant differences in the indicated mRNA levels between PR8 rp ‐infected ZAP‐KO cells and PR8 rp ‐infected Ctrl‐KO cells; pound symbols indicate statistically significant differences in the indicated mRNA levels between PR8‐infected ZAP‐KO cells and PR8‐infected Ctrl‐KO cells. ns, not significant; * or #, p < 0.05; ** or ##, p < 0.01; ***, p < 0.001; ****, p < 0.0001. C) Western blot analysis of viral proteins (PB2, HA, NP, NA, NS1) and ZAP isoforms (ZAP‐L, ZAP‐S) in lysates from PR8 rp ‐ or PR8‐infected ZAP‐KO and Ctrl‐KO cells at 24 hpi. GAPDH served as a loading control.

Article Snippet: The A/PR/8/34 (H1N1) (PR8) (VR‐95PQ, ATCC) and A/Aichi/2/68 (H3N2) (VR‐547, ATCC) were maintained in the laboratory.

Techniques: Gene Expression, CRISPR, Generated, Knock-Out, Control, Infection, Virus, Plaque Assay, Two Tailed Test, Western Blot