993. Combining Key Residues of the Russian and U.S. Live-Attenuated Influenza Viruses for a More Attenuated Virus.
Session: Poster Abstract Session: Adult and Pediatric Influenza Vaccine
Friday, October 5, 2018
Room: S Poster Hall
Posters
  • IDSA Conference Poster.pdf (385.0 kB)
  • 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.

    Minireplicon assay.

    minigenome%20methods.png

    Polymerase activity of combination mutants.

    Apsa%20Minigenomes.pdf

    Multicycle replication kinetics of combination mutants (Red) against PR8 Len (Black) at 33C (Solid), 37C (Dashed) and 39C (Dotted).Apsa%20Growth%20curves%20combo.pdf

    Andrew Smith, M.D./Ph.D. Candidate1, Laura Rodriguez-Garcia, Ph.D.1, Maya El Ghouayel, M.H.S., M.D. Candidate2, Luis Martinez-Sobrido, Ph.D.1 and Stephen Dewhurst, Ph.D.1, (1)Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY, (2)University of Rochester School of Medicine and Dentistry, Rochester, NY

    Disclosures:

    A. Smith, None

    L. Rodriguez-Garcia, None

    M. El Ghouayel, None

    L. Martinez-Sobrido, None

    S. Dewhurst, None

    Findings in the abstracts are embargoed until 12:01 a.m. PDT, Wednesday Oct. 3rd with the exception of research findings presented at the IDWeek press conferences.