Multiple amino acid residues confer temperature sensitivity to human influenza virus vaccine strains (flumist) derived from cold-adapted a/ann arbor/6/60

Abstract

FluMist influenza A vaccine strains contain the PB1, PB2, PA, NP, M, and NS gene segments of ca A/AA/6/60, the master donor virus-A strain. These gene segments impart the characteristic cold-adapted (ca), attenuated (att), and temperature-sensitive (ts) phenotypes to the vaccine strains. A plasmid-based reverse genetics system was used to create a series of recombinant hybrids between the isogenic non-ts wt A/Ann Arbor/6/60 and MDV-A strains to characterize the genetic basis of the ts phenotype, a critical, genetically stable, biological trait that contributes to the attenuation and safety of FluMist vaccines. PB1, PB2, and NP derived from MDV-A each expressed determinants of temperature sensitivity and the combination of all three gene segments was synergistic, resulting in expression of the characteristic MDV-A ts phenotype. Site-directed mutagenesis analysis mapped the MDV-A ts phenotype to the following four major loci: PB1(1195) (K391E), PB1(1766) (E581G), PB2(821) (N265S), and NP(146) (D34G). In addition, PB1(2005) (A661T) also contributed to the ts phenotype. The identification of multiple genetic loci that control the MDV-A ts phenotype provides a molecular basis for the observed genetic stability of FluMist vaccines.

Interferon-Induced Expression of MxA in the Respiratory Tract of Rhesus Macaques Is Suppressed by Influenza Virus Replication

“…This feature of influenza virus pathogenesis was also used to reduce the virulence of human influenza strains and produce a safe live influenza vaccine. The currently licensed, live-attenuated, cold-adapted influenza vaccine uses a master donor virus that is adapted for replication in primary chicken kidney cells at low temperature, producing a virus capable of only limited nonpathogenic infection of humans and limited transmission among humans (51). Ferrets are more susceptible to infection with human influenza isolates than mice and develop rhinitis that is similar to that in humans.…”

Towards an Epitope-Based Human Vaccine for Influenza

“…In the case of the attenuated live vaccines, the virus is cold adapted, e.g., attenuated by serial passage at low temperature, resulting in a live virus that can not cause significant illness in human. 1 In addition to the whole virus trivalent vaccine, subunit vaccines that are based on viral envelope proteins (HA and NA) are also approved for human use. It has been shown that such vaccines are highly immunogenic and well tolerated in children, young adults, and among the elderly.…”

Nasal-associated lymphoid tissues (NALTs) support the recall but not priming of influenza virus-specific cytotoxic T cells

“…Two types of influenza vaccines are currently available, the trivalent or quadrivalent inactivated influenza vaccine (TIV/QIV), which is given intramuscularly, and the live attenuated influenza vaccine (LAIV), which is administered intranasally. LAIV have been engineered by reverse genetics using the HA and NA genes from circulating viruses and an attenuated, temperature-sensitive, cold-adapted backbone, which prevents replication of the virus at temperatures above 33°C, thus limiting virus infection to the upper airways (8,9). Following inhalation of LAIV, the palatine tonsils, the NALT equivalent in humans, is the site where the humoral immune response is initiated (15).…”

“…We next assessed the anatomical location of CTL priming following immunization with the LAIV vaccine, which is engineered to preferentially replicate in the lower temperatures of the upper airways (7)(8)(9) and monitored the endogenous CD8 + T-cell response directed against influenza viral proteins. The CD8 + T-cell response following influenza virus infection of C57BL/6 mice is predominately directed against two viral proteins, the nucleoprotein (NP) and acid polymerase (PA) and can be monitored using H-2D b tetramers loaded with the PA 224 and NP 366 epitopes.…”