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Background The Live Attenuated Influenza Virus (LAIV) used in the United States 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 United States. 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 multicycle 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 33°C, 37°C, and 39°C 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 with WT PR8 at 37°C and 39°C 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 37°C and 39°C 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 37°C as compared with PR8 (Len) and an even greater decrease compared with PR8 (AA). Conclusion Our findings suggest combining the AA and Len LAIV polymerase mutations decreases LAIV replication at body temperature (37°C), as compared with 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. Minireplicon assay. Polymerase activity of combination mutants. Multicycle replication kinetics of combination mutants (red) against PR8 Len (black) at 33C (solid), 37C (dashed) and 39C (dotted). Disclosures All authors: No reported disclosures.

993. Combining Key Residues of the Russian and US Live-Attenuated Influenza Viruses for a More Attenuated Virus