Genetic stability and mutant selection in Sabin 2 strain of oral poliovirus vaccine grown under different cell culture conditions.
Mutations that consistently accumulated in the attenuated Sabin 2 strain of poliovirus during propagation in cell cultures were identified by sequence heterogeneity assay and quantified by mutant analysis by PCR and restriction enzyme cleavage (MAPREC). Eight additional sites previously identified in stool isolates were also examined by MAPREC in the virus passages. The pattern of selectable mutations and the rate of their accumulation depended on the type and confluence of the cell culture and the temperature of virus growth. Five unstable genomic sites were identified in Sabin 2 virus passaged 10 times at 34 degrees in African green monkey kidney (AGMK) cells, with the mutations accumulating in the range 1 to 24%. Accumulation of these mutations did not appear to result in a loss of attenuated phenotype since the virus passaged under these conditions passed the monkey neurovirulence test (MNVT). The content of the 481-G revertant known to be related to neurovirulence in monkeys did not increase. Thus, our results suggest that upon growth of Sabin 2 virus in AGMK cells at 34 degrees, the key determinant(s) of attenuation remained stable, and the mutations that occurred did not affect monkey neurovirulence. In virus passaged 10 times at 37 degrees in AGMK cells, 4 unstable genomic sites were identified, in some of them accumulating up to 12% of the mutants. This virus sample severely failed the MNVT. Virus passaged in Vero cells at 34 and 37 degrees accumulated mutants at 7 and 14 genomic sites, respectively, including 481-G in both cases, with almost complete substitution of the original nucleotides at some of the sites. We tested 44 commercial monopools of Type 2 OPV and found out that all of them contained 481-G revertants in the range 0.4-1.1%. An increase in the 481-G revertants in passaged viruses to the level of 4% and above correlated with failure of these samples by the MNVT. Since the pattern of selectable mutations differed in viruses grown in the two cell cultures used in this study, specific mutation profiles should be determined for each cell substrate used for vaccine production to assess manufacturing consistency.
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Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20852-1448.
Chumakov
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Rezapkin
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Lu
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Douthitt
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Dragunsky
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Levenbook
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers; D018076:DNA, Complementary; D004279:DNA, Viral / Q000235:genetics*; D007668:Kidney; D008969:Molecular Sequence Data; D017354:Point Mutation; D017955:Poliovirus / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity*; D011055:Poliovirus Vaccine, Oral; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D015183:Restriction Mapping; D014709:Vero Cells; D014774:Virulence
D017931:DNA Primers; D018076:DNA, Complementary; D004279:DNA, Viral; D011055:Poliovirus Vaccine, Oral; D012367:RNA, Viral