Optimizing the Live Attenuated Influenza A Vaccine Backbone for High-Risk Patient Groups

Figures

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  FIG 1. OptiLAIV NS1 binds to CPSF30 without compromising vaccine virus growth in eggs or MDCK cells. (A) 293T cells were transfected with pCAGGS.Flag-CPSF30 or pCAGGS.Flag-ZsGreen for 24 h prior to infection with either LAIV or optiLAIV at a multiplicity of infection (MOI) of 5. Cells were incubated for 16 h at 33°C and then lysed. Anti-Flag M2 affinity beads were used to immunoprecipitate (IP) the flag-tagged protein and its interactors. Precipitates were separated by SDS-PAGE and detected by Western blotting (WB) using the anti-flag antibody and the anti-NS1 antibody. The amount of NS1 protein that was bound to Flag-CPSF30 was quantified via a densiometric analysis (n = 2). (B) 10-day-old embryonated chicken eggs were infected with 100 PFU, and MDCK B cells were infected at a MOI of 0.1. After 46 h at 33°C, allantoic fluid and supernatant were collected. Viral titers were determined via plaque assay in MDCK cells at 33°C. Graphs represent the mean ± standard deviation (SD) of n = 3 to 7 repeats. Statistical significance was determined using a Mann-Whitney test.

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  FIG 2. OptiLAIV is attenuated and protects adult mice against a homosubtypic/heterologous challenge. (A) Scheme of mouse immunization and challenge. (B) Female 8-week-old mice were vaccinated intranasally under anesthesia with 104 PFU LAIV or optiLAIV in 25 μL PBS and body weight was monitored for 14 days postvaccination. (C) Female 8-week-old mice were vaccinated intranasally under anesthesia with 105 PFU LAIV or optiLAIV in 25 μL and at 4 days postvaccination snouts (left panel) and lungs (right panel) were collected and vaccine viral titters were determined by plaque assay (n = 7 per group). (D) Female 8-week-old mice vaccinated with 104 PFU of LAIV or optiLAIV were challenged at day 21 postvaccination with 20 PFU (10×LD50) of mouse adapted A/Netherlands/602/2009 (H1N1) in 20 μL PBS under light anesthesia (n = 14 per group). Body weight (left panel) and survival (right panel) were monitored for 14 days postchallenge. The statistical significances between LAIV group and optiLAIV group were determined using Mann-Whitney test in panel C; two-way ANOVA with the Geisser-Greenhouse correction and post hoc Dunn’s multiple-comparison test in panels B and D and Mantel-Cox test for survival…

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  FIG 3. OptiLAIV is attenuated and protects neonatal mice against homosubtypic/heterologous challenge and heterosubtypic challenge. (A) Scheme of mouse immunization and challenge. (B) Seven-day-old mice were vaccinated intranasally with 104 PFU LAIV or optiLAIV in 5 μL PBS and body weight was monitored for 14 days postvaccination. Cutoff was defined as twice the difference between the average weight of the nonvaccinated group and the lightest mouse of that group, calculated daily. (C) Seven-day-old mice were vaccinated intranasally with 105 PFU of LAIV or optiLAIV in 5 μL PBS. At 2, 4, 6, 8 and 9 days postvaccination vaccine viral titters in the snouts were determined by plaque assay (n = 11 to 19 per group). (D) Seven-day-old mice vaccinated with 104 PFU of LAIV or optiLAIV were challenged at day 21 postvaccination with 103 PFU (20×LD50) of A/Vietnam/1203/2004 (H5N1) in 20 μL PBS under anesthesia (n = 11 per group). Body weight (left panel) and survival (right panel) were monitored for 14 days postchallenge. (E) Seven-day-old mice vaccinated with 104 PFU of LAIV or optiLAIV were challenged at day 49 postvaccination with 20 PFU (10×LD50) of A/Netherlands/602/2009 (H1N1) in 20 μL PBS…

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  FIG 4. OptiLAIV is attenuated and safe in Stat1−/− mice. (A) Scheme of mouse immunization. (B) 8-week-old Stat1−/− mice were vaccinated intranasally under anesthesia with 104 PFU LAIV or optiLAIV in 25 μL and body weight (left panel) and survival (right panel) were monitored for 14 days postvaccination (n = 4 mock-vaccinated group; n = 8 per vaccinated group). (C) 8-week-old Stat1−/− mice were vaccinated intranasally under anesthesia with 105 PFU LAIV or optiLAIV in 25 μL PBS. At 2, 4 and 6 days postvaccination snouts (left panel) and lungs (right panel) were collected and viral titers were determined by plaque assay (n = 5 per group). The statistical significances between LAIV group and optiLAIV group were determined using two-way ANOVA with the Geisser-Greenhouse correction and post hoc Dunn’s multiple-comparison test in panel B and Mantel-Cox test for survival curve; multiple Mann-Whitney tests in panel C;. P-values are indicated in the figure. *, comparison between LAIV and optiLAIV. L.o.D. = limit of detection (5 PFU/mL). Graphs are representative of 2 independent experiments and indicate mean ± SEM for panel B and geometric mean for panel C. Symbols represent data from individual…

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  FIG 5. OptiLAIV NS1 allows unfolded protein response activation in human cell models. (A–B) A549 cells were infected at an MOI of 5 with LAIV or optiLAIV or treated with tunicamycin at 5 μg/mL. At 16 h and 24 h postinfection RT-PCR was performed from isolated RNA for the spliced form of XBP1 mRNA (A). In parallel at 8 h, 16 h and 24 h postinfection, lysates were analyzed by Western blot for expression of sXBP1, actin, NP and NS1 proteins (B). (C) A549wt cells (upper panel) or A549Stat1−/− cells (lower panel) were infected at an MOI of 5 with LAIV or optiLAIV and RT-qPCR for UPR-induced genes DNAJB9 and DDIT3 was performed in RNA lysates at 16 h and 24 h postinfection. (D) A549wt cells (upper panel) or A549Stat1−/− cells (lower panel) were infected at an MOI of 0.01 with either LAIV or optiLAIV. Supernatants were collected at indicated time postinfection and viral titers determined by plaque assay. (E) Primary human nasal epithelial cells (Mucilair) were infected at an MOI of 5 with LAIV or optiLAIV. Cells were lysed at 24 h postinfection and RT-qPCR for each respective gene was performed. The statistical significances between LAIV group and optiLAIV group were determined using one-way…