Journal: Journal of Virology

Article Title: Re-engineering segment 8 facilitates generation of a versatile live-attenuated influenza A virus vector platform for secretory protein delivery

doi: 10.1128/jvi.00347-26

Figure Lengend Snippet: Re-engineered segment 8 enables seamless integration of the human IL-2 ORF into the IAV-ΔNS1 vector backbone. ( a ) Schematic representation of alternative splicing for influenza A virus mRNA encoded by wild-type or re-engineered segment 8. Icon descriptions are provided in the figure. Not to scale. ( b ) Representative agarose gel image showing segment 8-specific RT-PCR products amplified from viral RNAs for the indicated viruses. ( c ) Ratios of spliced versus total segment 8 mRNA copy numbers from HEK293T cells infected with viruses carrying wild-type or ΔNS1-IL-2 segment 8 at an MOI of 1, quantified by qRT-PCR, at the indicated time points. Representative data from two independent experiments are depicted as mean ± SD ( n = 3). ( d ) Western blot images acquired from lysates of HEK293T cells infected with the indicated viruses or mock controls at 24 hours post-infection. A representative image is shown. ( e ) Multi-cycle growth curve analysis for the indicated viruses in MDCK-NS1 cells. Data are depicted as mean ± SD ( n = 3). Representative data from two independent experiments are shown. * and # indicate statistically significant differences between wild-type and vector control, or between wild-type and the IL-2-carrying vector, respectively. IL-2 ELISA ( f ) and IL-2 bioactivity assay ( g ) results for supernatants collected from MDCK-NS1 cells infected with indicated viruses at an MOI of 1 at 24 hpi. Representative data from two independent experiments are depicted as mean ± SD ( n = 3). ( h ) IL-2 ELISA results for supernatants collected from consecutive blind passages of ∆NS1-IL-2 viruses on MDCK-NS1 cells. Data are depicted as mean ± SD ( n = 3). Student’s t -test was used to test for statistical significance unless otherwise stated. P < 0.05. bp, base pair; hpi, hours post-infection; IB, immunoblot; kDa, kilodalton; pfu, plaque-forming unit; mL, milliliter; and ng, nanogram.

Article Snippet: The human IL-2 gene, amplified from the commercially available plasmid (RC210013, Origene) and flanked with homologous sequences, was fused to the adjacent segments by nested PCR and cloned into a pDZ vector.

Techniques: Plasmid Preparation, Alternative Splicing, Virus, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Amplification, Infection, Quantitative RT-PCR, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of Virology

Article Title: Re-engineering segment 8 facilitates generation of a versatile live-attenuated influenza A virus vector platform for secretory protein delivery

doi: 10.1128/jvi.00347-26

Figure Lengend Snippet: Novel IAV-ΔNS1 vectors induce notable viral antigen expression and vector-sourced IL-2 secretion in IFN-competent systems. ( a ) Representative images of plaque assays for pre-estimated doses of indicated viruses at 33°C or 37°C on MDCK or MDCK-NS1 cells. Plaques were stained by standard immuno-staining against IAV NP protein. ( b ) Multi-cycle growth curve analysis for the indicated viruses in MDCK cells. Data are depicted as mean ± SD ( n = 3). Representative data from two independent experiments are shown. * and # indicate statistically significant differences between wild-type and vector control, or between wild-type and the IL-2-carrying vector, respectively. ( c and d ) MDCK, A549, and HEK293T cells were infected with the indicated viruses at an MOI of 1. ( c ) At 24 hpi, infection rates were quantified by flow cytometry analysis for IAV NP-expressing cells. Representative data from two independent experiments are depicted as mean ± SD ( n = 3). One-way ANOVA was applied to test for statistical significance. P < 0.05. ( d ) IL-2 levels in culture supernatants and bioactivity of vector-sourced IL-2 were measured at the indicated time points by sandwich ELISA and HEK-Blue CD122/CD132 cells, respectively. Representative data from two independent experiments are depicted as median ( n = 3). ( e and f ) BALB/c mice were intranasally inoculated with the vectors at a dose of 1 × 10 5 pfu/animal or mock controls. ( e ) Animals were monitored for weight loss up to 7 days post-infection ( n = 5). The dotted line indicates the corresponding human endpoint (75% of initial body weight). Data are depicted as mean ± SD. ( f ) IL-2 levels measured by sandwich ELISA in bronchoalveolar lavages (BALs) and serum collected from mice inoculated with the indicated viruses or mock controls at 24 hpi. Data are depicted as mean ± SD. Student’s t -test was used to test for statistical significance unless otherwise stated. P < 0.05. ng, nanogram; mL, milliliter; and hpi, hours post-infection.

Article Snippet: The human IL-2 gene, amplified from the commercially available plasmid (RC210013, Origene) and flanked with homologous sequences, was fused to the adjacent segments by nested PCR and cloned into a pDZ vector.

Techniques: Expressing, Plasmid Preparation, Staining, Immunostaining, Control, Infection, Flow Cytometry, Sandwich ELISA

Journal: Journal of Virology

Article Title: Re-engineering segment 8 facilitates generation of a versatile live-attenuated influenza A virus vector platform for secretory protein delivery

doi: 10.1128/jvi.00347-26

Figure Lengend Snippet: Re-engineered segment 8 enables seamless integration of the human IL-2 ORF into the IAV-ΔNS1 vector backbone. ( a ) Schematic representation of alternative splicing for influenza A virus mRNA encoded by wild-type or re-engineered segment 8. Icon descriptions are provided in the figure. Not to scale. ( b ) Representative agarose gel image showing segment 8-specific RT-PCR products amplified from viral RNAs for the indicated viruses. ( c ) Ratios of spliced versus total segment 8 mRNA copy numbers from HEK293T cells infected with viruses carrying wild-type or ΔNS1-IL-2 segment 8 at an MOI of 1, quantified by qRT-PCR, at the indicated time points. Representative data from two independent experiments are depicted as mean ± SD ( n = 3). ( d ) Western blot images acquired from lysates of HEK293T cells infected with the indicated viruses or mock controls at 24 hours post-infection. A representative image is shown. ( e ) Multi-cycle growth curve analysis for the indicated viruses in MDCK-NS1 cells. Data are depicted as mean ± SD ( n = 3). Representative data from two independent experiments are shown. * and # indicate statistically significant differences between wild-type and vector control, or between wild-type and the IL-2-carrying vector, respectively. IL-2 ELISA ( f ) and IL-2 bioactivity assay ( g ) results for supernatants collected from MDCK-NS1 cells infected with indicated viruses at an MOI of 1 at 24 hpi. Representative data from two independent experiments are depicted as mean ± SD ( n = 3). ( h ) IL-2 ELISA results for supernatants collected from consecutive blind passages of ∆NS1-IL-2 viruses on MDCK-NS1 cells. Data are depicted as mean ± SD ( n = 3). Student’s t -test was used to test for statistical significance unless otherwise stated. P < 0.05. bp, base pair; hpi, hours post-infection; IB, immunoblot; kDa, kilodalton; pfu, plaque-forming unit; mL, milliliter; and ng, nanogram.

Article Snippet: Human IL-2 ( NM_000586 ) and IL-15 ( NM_172174 ) complementary DNAs (cDNAs) were cloned from commercially available plasmids (RC210013 and RC219294, respectively; Origene).

Techniques: Plasmid Preparation, Alternative Splicing, Virus, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Amplification, Infection, Quantitative RT-PCR, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of Virology

Article Title: Re-engineering segment 8 facilitates generation of a versatile live-attenuated influenza A virus vector platform for secretory protein delivery

doi: 10.1128/jvi.00347-26

Figure Lengend Snippet: Novel IAV-ΔNS1 vectors induce notable viral antigen expression and vector-sourced IL-2 secretion in IFN-competent systems. ( a ) Representative images of plaque assays for pre-estimated doses of indicated viruses at 33°C or 37°C on MDCK or MDCK-NS1 cells. Plaques were stained by standard immuno-staining against IAV NP protein. ( b ) Multi-cycle growth curve analysis for the indicated viruses in MDCK cells. Data are depicted as mean ± SD ( n = 3). Representative data from two independent experiments are shown. * and # indicate statistically significant differences between wild-type and vector control, or between wild-type and the IL-2-carrying vector, respectively. ( c and d ) MDCK, A549, and HEK293T cells were infected with the indicated viruses at an MOI of 1. ( c ) At 24 hpi, infection rates were quantified by flow cytometry analysis for IAV NP-expressing cells. Representative data from two independent experiments are depicted as mean ± SD ( n = 3). One-way ANOVA was applied to test for statistical significance. P < 0.05. ( d ) IL-2 levels in culture supernatants and bioactivity of vector-sourced IL-2 were measured at the indicated time points by sandwich ELISA and HEK-Blue CD122/CD132 cells, respectively. Representative data from two independent experiments are depicted as median ( n = 3). ( e and f ) BALB/c mice were intranasally inoculated with the vectors at a dose of 1 × 10 5 pfu/animal or mock controls. ( e ) Animals were monitored for weight loss up to 7 days post-infection ( n = 5). The dotted line indicates the corresponding human endpoint (75% of initial body weight). Data are depicted as mean ± SD. ( f ) IL-2 levels measured by sandwich ELISA in bronchoalveolar lavages (BALs) and serum collected from mice inoculated with the indicated viruses or mock controls at 24 hpi. Data are depicted as mean ± SD. Student’s t -test was used to test for statistical significance unless otherwise stated. P < 0.05. ng, nanogram; mL, milliliter; and hpi, hours post-infection.

Article Snippet: Human IL-2 ( NM_000586 ) and IL-15 ( NM_172174 ) complementary DNAs (cDNAs) were cloned from commercially available plasmids (RC210013 and RC219294, respectively; Origene).

Techniques: Expressing, Plasmid Preparation, Staining, Immunostaining, Control, Infection, Flow Cytometry, Sandwich ELISA