h1n1 pr8  (ATCC)

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

Journal: mBio

Article Title: An NLRP3-stimulatory adjuvant improves the immunogenicity of influenza virus vaccines in mice and non-human primates

doi: 10.1128/mbio.02343-25

Figure Lengend Snippet: Including R848 and CT009 in vaccinations protects mice from lethal PR8 influenza challenge. ( A ) Timeline of study shows two vaccine doses preceding lethal dose infection of BALB/c mice with 1,000 plaque-forming unit (PFU) of PR8 virus. Mice were monitored for ( B ) body weight changes; ( C ) disease symptoms; ( D ) survival. For survival data, significance was determined compared to PBS. A cohort of mice was sacrificed on day 4 post-infection for assessment of ( E ) HA-specific antibodies; ( F ) NP-specific CD8 T cells in draining lymph nodes; ( G ) IFNγ secretion from splenocytes after restimulation with IAV. Means and SD are shown. Each symbol in the bar graph represents one mouse; n = 5–8 mice per group. Significance was determined by log-rank Mantel-Cox test ( D ), Two-way ANOVA followed by Dunnett’s multiple comparison ( E ) or one-way ANOVA followed by Tukey’s multiple comparisons ( F–G ). * P < 0.05; **** P < 0.0001.

Article Snippet: Influenza virus (H1N1) strain A/PR/8/34 (PR8) was purchased from ATCC and propagated in MDCK cells at 37°C for 2 days.

Techniques: Infection, Virus, Comparison