Background: The Live Attenuated Influenza Virus (LAIV) used in the US is based on the cold-passaged A/AnnArbor/6/60 strain (AA). An alternative LAIV (Len), developed from the cold-passaged A/Leningrad/134/17/57 strain, has also been used in some countries outside the US. Recent concerns with the efficacy and safety of the current US LAIV warrant the development of an improved LAIV.
Methods: We used in vitro minireplicon and multi-cycle viral growth assays to analyze the combined effects of polymerase mutations from LAIV (AA) and LAIV (Len) on the phenotype of PR8. Mini-replicon assays were performed in HEK-293T cells with firefly luciferase under the control of the influenza virus NP promoter; we controlled for cell density with a constitutively-active Renilla luciferase. Multicycle growth curve experiments were performed at 33C, 37C, and 39C in MDCK cells with an m.o.i. of 0.001. Mean values for triplicate infections at 12, 24, 48, and 72 hours were plotted as TCID50/mL.
Results: Control experiments showed replication of PR8 (AA) and PR8 (Len) in MDCK cells was significantly decreased as compared to WT PR8 at 37C and 39C at 24-48 hour time points, but not at 33C (the temperature of nasal passages). We found that polymerase activity was up to 3 logs more temperature-sensitive (ts) at 37C and 39C with the combined Len and AA mutations using the mini-replicon assay. In the growth curve experiments, the combined Len and AA mutations conferred up to a 4-log decrease in replication levels at 37C as compared to PR8 (Len) and an even greater decrease compared to PR8 (AA).
Conclusion: Our findings suggest combining the AA and Len LAIV polymerase mutations decreases LAIV replication at body temperature (37C), as compared to either LAIV alone. This could be useful in developing an improved LAIV that is safer in vulnerable hosts (e.g., children under the age of 2 who may be vulnerable to wheezing), while also permitting dose escalation that might result in greater efficacy.
Polymerase activity of combination mutants.
M. El Ghouayel, None
L. Martinez-Sobrido, None
S. Dewhurst, None