Japanese encephalitis virus live attenuated vaccine strains display altered immunogenicity, virulence and genetic diversity

Abstract

Japanese encephalitis virus (JEV) is the etiological agent of Japanese encephalitis (JE). The most commonly used vaccine used to prevent JE is the live-attenuated strain SA14-14-2, which was generated by serial passage of the wild-type (WT) JEV strain SA14. Two other vaccine candidates, SA14-5-3 and SA14-2-8 were derived from SA14. Both were shown to be attenuated but lacked sufficient immunogenicity to be considered effective vaccines. To better contrast the SA14-14-2 vaccine with its less-immunogenic counterparts, genetic diversity, ribavirin sensitivity, mouse virulence and mouse immunogenicity of the three vaccines were investigated. Next generation sequencing demonstrated that SA14-14-2 was significantly more diverse than both SA14-5-3 and SA14-2-8, and was slightly less diverse than WT SA14. Notably, WT SA14 had unpredictable levels of diversity across its genome whereas SA14-14-2 is highly diverse, but genetic diversity is not random, rather the virus only tolerates variability at certain residues. Using Ribavirin sensitivity in vitro, it was found that SA14-14-2 has a lower fidelity replication complex compared to SA14-5-3 and SA14-2-8. Mouse virulence studies showed that SA14-2-8 was the most virulent of the three vaccine strains while SA14-14-2 had the most favorable combination of safety (virulence) and immunogenicity for all vaccines tested. SA14-14-2 contains genetic diversity and sensitivity to the antiviral Ribavirin similar to WT parent SA14, and this genetic diversity likely explains the (1) differences in genomic sequences reported for SA14-14-2 and (2) the encoding of major attenuation determinants by the viral E protein.

Fig. 1. Derivation of JEV vaccine strains.

The successful SA14-14-2 vaccine strain was derived after multiple attempts to attenuate WT strain SA14. The first of these attempts, Clone12-1-7, was generated by mouse and cell culture passage. It was then UV irradiated to generate SA14-2-8 and further mouse passaged to generate SA14-5-3. Both of these vaccine strains were shown to be overly attenuated in humans so SA14-5-3 was passaged in suckling mice which generated the currently utilized SA14-14-2.

Fig. 2. Consensus changes during JEV vaccine derivation.

The complete genomes of SA14, Clone12-1-7, SA14-2-8, SA14-5-3 and SA14-14-2 were compared. Consensus changes that represent a reversion to the parental, WT strain SA14 are bolded. Consensus changes that occur within the protein coding region of the genome are reported as the codon number within the gene. Consensus changes that occur outside the protein coding region are italicized and reported as a nucleotide number. Sequences of SA14, SA14-2-8, SA14-5-3 and SA14-14-2 were generated in house through Illumina sequencing. The sequence for Clone-12-1-7 was accessed from GenBank (AF416457).

Fig. 3. Genetic diversity of JEV WT and vaccine strains upon passage.

The genetic diversity of two sequential passages of SA14 and the three vaccine strains derived from it was compared through Illumina sequencing methods and quantified using Shannon entropy. The Shannon entropy of SA14 (a), SA14-14-2 (b), SA14-5-3 (c) and SA14-2-8 (d) were mapped across the genome. All nucleotide positions, UTRs included, are depicted.

Fig. 4. Genetic diversity of JEV vaccine strains compared to parental WT strain.

The genetic diversity of SA14 and the three vaccine strains derived from it was compared through Illumina sequencing methods and quantified using Shannon entropy (a). The Shannon entropy of SA14 (b), SA14-14-2 (c), SA14-5-3 (d) and SA14-2-8 (e) were mapped across the genome. All nucleotide positions, UTRs included, are depicted in (a): *p = 0.014 (SA14 vs SA14-14-2), **p = 0.0003 (SA14-14-2 vs SA14-5-3), ***p < 0.0001 (SA14-14-2 vs SA14-2-8).

Fig. 5. Frequency and genome location of single nucleotide variants detected in SA14 and vaccine derivatives.

RNA was extracted and sequenced using Illumina methods. LoFreq software was used to detect SNVs in SA14 (A), SA14-14-2 (B), SA14-5-3 (C) and SA14-2-8 (D) samples. Dotted lines represent 50% of the population at which an SNV would become the consensus nucleotide and 1% of the population. An SNV frequency cutoff was applied at 0.05% frequency.

Fig. 6. Dose response of WT JEV and JEV vaccine strains to the antiviral ribavirin.

SA14, SA14-14-2, SA14-5-3 and SA14-2-8 were incubated with the GTP nucleoside analog, ribavirin. After 48 h, the supernatant was collected and titrated for viral load (FFU). Titers at each concentration were normalized to untreated, infected cells and fit using a dose-response linear regression. The experiment was undertaken in triplicate and points shown are an average of these experiments. A four-parameter, non-linear regression was used to fit sensitivity curves (SA14 R²= 0.89, SA14-14-2 R²= 0.89, SA14-5-3 R²= 0.79, SA14-2-8 R²= 0.964).

Fig. 7. Summary of attenuating E protein mutations in JEV vaccine strains.

The three attenuated vaccine strains were empirically derived which led to distinct combinations of genotypes and phenotypes (A). It is apparent that genetic diversity doesn’t correlate with attenuation in mice. It is proposed that changes to the E protein of SA14-2-8 (shown in green) and SA14-14-2 (shown in blue) are responsible for differences in mouse virulence (B).