Genetic bases of the temperature-sensitive phenotype of a master donor virus used in live attenuated influenza vaccines: A/Leningrad/134/17/57 (H2N2)

Abstract

Trivalent live attenuated influenza vaccines whose type A components are based on cold-adapted A/Leningrad/134/17/57 (H2N2) (caLen17) master donor virus (MDV) have been successfully used in Russia for decades to control influenza. The vaccine virus comprises hemagglutinin and neuraminidase genes from the circulating viruses and the remaining six genes from the MDV. The latter confer temperature-sensitive (ts) and attenuated (att) phenotypes. The ts phenotype of the vaccine virus is a critical biological determinant of attenuation of virulence. We developed a plasmid-based reverse genetics system for MDV caLen17 to study the genetic basis of its ts phenotype. Mutations in the polymerase proteins PB1 and PB2 played a crucial role in the ts phenotype of MDV caLen17. In addition, we show that caLen17-specific ts mutations could impart the ts phenotype to the divergent PR8 virus, suggesting the feasibility of transferring the ts phenotype to new viruses of interest for vaccine development.

Characterization of Reverse Genetics-Derived Cold-Adapted Master Donor Virus A/Leningrad/134/17/57 (H2N2) and Reassortants with H5N1 Surface Genes in a Mouse Model

“…The internal genes that include the ca mutations were also major determinants of the phenotypic characteristics of caVNLen17rg compared to VN-Len134rg parent virus, in agreement with the in vitro findings (37). Reversion of nine amino acid mutations from the cold-adapted virus dramatically shifted virus tro- 50 of either VN1203 or EG321 HPAI virus.…”

“…with wt Len134 had ϳ4.5 log 10 50% egg infective doses (EID 50 ) in lungs at 3 dpi, whereas the caLen17 virus was below detection levels. We generated reverse genetics-derived Len134 virus (37), but despite similar temperature-sensitive profiles of Len134 and Len134-rg viruses in MDCK cells (Fig. 1), the latter virus replicated in mouse lung significantly less efficiently than did the authentic Len134 virus.…”

“…Len134-rg virus was derived by reverting caLen17-specific mutations by reverse genetics (rg) as described previously (37). Site-directed mutagenesis of the plasmids was performed using the QuikChange XL multisite-directed mutagenesis kit (Stratagene, La Jolla, CA).…”

“…H5N1 reassortants, as well as wild-type H2N2 viruses, were handled in biosafety level 3 containment facilities. Virus titers were determined by plaque assay in MDCK cells as described previously (37) and expressed in log 10 PFU/ml. MID 50 determination.…”

Reassortant viruses for influenza vaccines: is it time to reconsider their genome structures?

“…Hence, it will be important to ensure that replacement of the NP or M gene in the genome of LAIV viruses does not affect their temperature sensitivity. The molecular signatures of the ts phenotypes of A/Ann Arbor/1/66 and A/Leningrad/134/17/57-based LAIV strains have been intensively studied, and the phenotype has been mapped to mutations in the polymerase proteins PB2 and PB1 [17,18]. In addition, a mutation in NP (D34G) has been identified as one of the determinants of temperature sensitivity of the A/Ann Arbor/6/66 MDV [17].…”