Genetic Stability and Mutant Selection in Sabin 2 Strain of Oral Poliovirus Vaccine Grown under Different Cell Culture Conditions

Abstract

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.

Molecular Basis for Attenuation of Neurovirulence of a Yellow Fever Virus/Japanese Encephalitis Virus Chimera Vaccine (ChimeriVax-JE)

“…Despite this, it is known that as few as two mutations in the E protein of the YFV 17D vaccine can revert the virus to a neurovirulent phenotype, although this is a very rare occurrence (22). This points out the fact that absolute stabilization of the vaccine phenotype in a cell culture-passaged virus is very problematic, a reality that has been faced for other live-attenuated viral vaccines such as poliovirus (45,54). To address the question of whether single or multiple amino acid reversions in the E protein of ChimeriVax-JE were required to increase its neurovirulence properties, we tested viruses which contained substitutions at sites known to be associated with the attenuation process.…”

Modulation of Poliovirus Replicative Fitness in HeLa Cells by Deoptimization of Synonymous Codon Usage in the Capsid Region

“…Apart from the back mutation at position 3120, all other variable sites differed between ABCD, ABCd, and abcd. No net changes were observed at sites A 481 (in the 5Ј UTR) and U 2909 (in the VP1 region), which are known to be subject to strong negative selection when Sabin 2 replicates in the human intestine (44,49,78,79), and no changes were found at other sites known to vary upon propagation of Sabin 2 in cell culture (69).…”

Long-Term Circulation of Vaccine-Derived Poliovirus That Causes Paralytic Disease

“…In short, DNA copies of segments of the VP1 (nucleotides [nt] 2402 to 2881; numbering of the Sabin 1 RNA), 2AB (nt 3617 to 4152), 2C (nt 4169 to 4965), and 3D (nt 6086 to 6376) genomic regions were amplified by PCR with appropriate primers; the samples were separately treated with restriction endonucleases HaeIII, HinfI, HpaII, DdeI, and RsaI; and fragments were analyzed by 3% agarose gel electrophoresis at 5 V/cm for 2 h and visualized by ethidium bromide staining. For determination of complete genome sequences, DNA copies of 11 genomic fragments were amplified by PCR (57,64), and the PCR products were purified with the QIAquick system (Qiagen) and sequenced using an ABI Prism 310 Genetic Analyzer (Applied Biosystems) as specified by the manufacturer. Nucleotides at each position of the genome were determined at least twice by reading both complementary strands.…”

“…For comparative analysis, we used the genome sequences of Sabin 1 and Sabin 2 strains as determined by Toyoda et al (70) and as corrected by direct sequencing of PCR products (57,64). Multiple alignment of the sequences determined here and those of Sabin strains was carried out with the program CLUSTAL W, version 1.74 (67).…”