Attenuation of the recombinant human parainfluenza virus type 3 cp45 candidate vaccine virus is augmented by importation of the respiratory syncytial virus 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 attenuation (att) phenotypes. Sequence alignment of this region in the L proteins of several distantly related paramyxoviruses revealed that this phenylalanine is conserved. Using reverse genetics, the analogous phenylalanine at position 456 in the L protein of wild-type PIV3 was mutagenized to leucine (F456L). The resulting virus, designated r456(L), was ts (40 degrees C shut-off temperature of plaque formation), and its replication in the upper, but not the lower, respiratory tract of hamsters was 10-fold reduced compared with that of the recombinant wild-type PIV3 (rwt). Thus the phenylalanine 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 rPIV3 candidate vaccine viruses, one bearing the three cp45 ts missense mutations in the L protein (rcp45(L)) and the other bearing all 15 cp45 mutations (rcp45), would further attenuate the viruses in vivo. Each rcp45 derivative to which the F456L mutation was added exhibited an increased level of temperature sensitivity. Furthermore rcp45(L)-456 and rcp45-456 were 100- to 1000-fold more restricted in replication in hamsters than their rcp45(L) and rcp45 parents. Despite the high level of restriction of replication in hamsters, 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 restricted in the respiratory tract of chimpanzees than rcp45 and induced a comparable, moderate to high level of PIV3-specific serum antibodies. rcp45 and rcp45-456 viruses isolated from chimpanzees throughout the 2-week course of replication maintained the level of temperature sensitivity of their respective input viruses, illustrating their phenotypic stability. Thus the acquisition of the F456L mutation by the cp45 virus resulted in a small, incremental increase in its level of attenuation, indicating its possible usefulness in the fine tuning of the level of attenuation of the cp45 vaccine candidate. The ability to transfer mutations identified in heterologous paramyxoviruses, which in this case represent different subfamilies, greatly enhances our ability to rapidly develop novel parainfluenza virus candidate vaccines.
Laboratory of Infectious Diseases, National Institutes of Health, Bethesda, Maryland, 20892-0720, USA. mskiadopoulos@atlas.niaid.nih.giv
M H
MH
Skiadopoulos
S R
SR
Surman
M
M
St Claire
W R
WR
Elkins
P L
PL
Collins
B R
BR
Murphy
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D006224:Cricetinae; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D002679:Pan troglodytes; D010224:Parainfluenza Virus 3, Human / Q000235:genetics / Q000276:immunology* / Q000502:physiology; D010641:Phenotype; D011994:Recombinant Proteins; D012136:Respiratory Syncytial Viruses / Q000235:genetics; D013696:Temperature; D014613:Vaccines, Attenuated; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D014765:Viral Vaccines; D014779:Virus Replication
D011994:Recombinant Proteins; D014613:Vaccines, Attenuated; D014764:Viral Proteins; D014765:Viral Vaccines; C056831:parainfluenza 3 virus, L Protein; D012321:DNA-Directed RNA Polymerases