Attenuation of the recombinant human parainfluenza virus type 3 cp45 candidate vaccine virus is augmented by importation of the respiratory syncytialvirus cpts530 L polymerase mutation

A phenylalanine to leucine mutation at position 521 in the L polymerase of cpts530, a live-attenuated respiratory syncytial virus (RSV) cold-passaged (cp), temperature-sensitive (ts) candidate vaccine, specifies the ts and at tenuation (att) phenotypes. Sequence alignment of this region in the L prot eins of several distantly related paramyxoviruses revealed that this phenyl alanine is conserved. Using reverse genetics, the analogous phenylalanine a t position 456 in the L protein of wild-type PIV3 was mutagenized to leucin e (F456L). The resulting virus, designated r456(L), was ts (40 degrees C sh ut-off temperature of plaque formation), and its replication in the upper, but not the lower, respiratory tract of hamsters was 10-Told reduced compar ed with that of the recombinant wild-type PIV3 (Mrt). Thus the phenylalanin e to leucine mutation specified a similar level of temperature sensitivity and attenuation in two distantly related paramyxoviruses. We next sought to determine whether the addition of this mutation to the L protein of two rP IV3 candidate vaccine viruses, one bearing the three cp45 ts missense mutat ions in the L protein (rcp45(L)) and the other bearing all 15 cp45 mutation s (rcp45), would further attenuate the viruses in viva. Each rcp45 derivati ve to which the F456L mutation was added exhibited an increased level of te mperature sensitivity. Furthermore rcp45(L)-456 and rcp45-456 were 100- to 1000-fold more restricted in replication in hamsters than their rcp45, and rcp45 parents. Despite the high level of restriction of replication in hams ters, immunization with rcp45-456 induced a moderate level of resistance to replication of PIV3 challenge virus. In contrast to the highly restricted replication observed in hamsters, rcp45-456 was only fivefold more restrict ed in the respiratory tract of chimpanzees than rcp45 and induced a compara ble, moderate to high level of PIV3-specific serum antibodies, rcp45 and rc p45-456 viruses isolated from chimpanzees throughout the 2-week course of r eplication maintained the level of temperature sensitivity of their respect ive input viruses, illustrating their phenotypic stability. Thus the acquis ition of the F456L mutation by the cp45 virus resulted in a small, incremen tal increase in its level of attenuation, indicating its possible usefulnes s in the fine tuning of the level of attenuation of the cp45 vaccine candid ate. The ability to transfer mutations identified in heterologous paramyxov iruses, which in this case represent different subfamilies, greatly enhance s our ability to rapidly develop novel parainfluenza virus candidate vaccin es. (C) 1999 Academic Press.