Analysis By Deep Sequencing of Discontinued Neurotropic Yellow Fever Vaccine Strains

Abstract

Deep sequencing of live-attenuated viral vaccines has focused on vaccines in current use. Here we report characterization of a discontinued live yellow fever (YF) vaccine associated with severe adverse events. The French neurotropic vaccine (FNV) strain of YF virus was derived empirically in 1930 by 260 passages of wild-type French viscerotropic virus (FVV) in mouse brain. The vaccine was administered extensively in French-speaking Africa until discontinuation in 1982, due to high rates of post-vaccination encephalitis in children. Using rare archive strains of FNV, viral RNAs were sequenced and analyzed by massively parallel, in silico methods. Diversity and specific population structures were compared in reference to the wild-type parental strain FVV, and between the vaccine strains themselves. Lower abundance of polymorphism content was observed for FNV strains relative to FVV. Although the vaccines were of lower diversity than FVV, heterogeneity between the vaccines was observed. Reversion to wild-type identity was variably observed in the FNV strains. Specific population structures were recovered from vaccines with neurotropic properties; loss of neurotropism in mice was associated with abundance of wild-type RNA populations. The analysis provides novel sequence evidence that FNV is genetically unstable, and that adaptation of FNV contributed to the neurotropic adverse phenotype.

Figure 1

Diversity (normalized Shannon’s entropy - NSE) and genetic distance (Root-mean squared distance - RMSD) from FVV were measured for all nucleotide positions in the single, open reading frame (ORF) of the YFV genomes. Vaccine strains were of lower diversity than wild-type parental strains, especially when exclusively considering nucleotide positions encoding amino acid substitutions that define the vaccine genotypes of FNV or 17D-204. All FNV vaccine strains were of lower diversity than the parental strain FVV, and likewise were of greater genetic distance from FVV. FNV-IP and FNV-Yale were of lowest diversity, whereas FNV-FC and FNC-NT were not stably fixed at the putative vaccine genotype (Table 2). (A) Sequencing read coverage for all viruses sequenced in the study, after random downsampling to normalize. (B) Paired comparison of NSE for Asibi and 17D-204, for the 20 nucleotide positions that encode amino acid substitutions observed in 17D vaccine substrains. This is provided as a method control to replicate the low-diversity pattern shown previously for 17D-204. (C) NSE for all nucleotide positions in viral ORFs. (D) RMSD relative to FVV for all nucleotide positions in viral ORFs. (E) NSE for 26 nucleotide positions encoding amino acid substitutions relative to FVV in at least 3 of 4 FNV strains. (F) RMSD for 26 nucleotide positions depicted in subfigure E.

Figure 2

Pairwise quantile-quantile plots of sequencing error rate for FVV and FNV strains. In this case, error rate contains both true variant populations and sequencing artifacts. The presence of non-consensus variant frequencies is observed by aggregation of points away from a slope of 1. P-values were generated by a bootstrapped Kolmogorov-Smirnov test, using a Bonferroni-corrected alpha of 0.008. Under these criteria, all pairings differ significantly except those of FNV-NT/FNV-Yale and FNV-NT/FNV-IP (*non-significant); this supports other observations in the study that FNV-NT and either FNV-Yale/IP are highly divergent in their subpopulations.

Figure 3

Summary of single-nucleotide polymorphism (SNP) content for YFV strains considered in the study. (A) SNPs were modeled using V-Phaser v.2.0; significantly represented variants were mapped to the consensus sequence of the virus from which they were detected. Variants are depicted along the YFV genome with respect to their frequency in the alignment, with gene boundaries shown; those generating an amino acid substitution are shown by symbol. SNPs encoding reversion to the wild-type (FVV or Asibi for 17D-204) genome are shown as vertical lines. (B) Plots of density for detected variants that code for amino acid substitutions, separated by wild-type revertant identity. Viruses with homogenous population structure (17D-204, FNV-Yale, FNV-IP) cluster at the lower end of the frequency range, whereas the less homogenous viruses (FNV-NT, FNV-FC) show density of counts at relatively higher ranges, with greater relative density of high-frequency revertants. Median count value is depicted by a vertical line.