Single mutation in the flavivirus envelope protein hinge region increases neurovirulence for mice and monkeys but decreases viscerotropism for monkeys: relevance to development and safety testing of live, attenuated vaccines

Abstract

A chimeric yellow fever (YF) virus/Japanese encephalitis (JE) virus vaccine (ChimeriVax-JE) was constructed by insertion of the prM-E genes from the attenuated JE virus SA14-14-2 vaccine strain into a full-length cDNA clone of YF 17D virus. Passage in fetal rhesus lung (FRhL) cells led to the emergence of a small-plaque virus containing a single Met-->Lys amino acid mutation at E279, reverting this residue from the SA14-14-2 to the wild-type amino acid. A similar virus was also constructed by site-directed mutagenesis (J. Arroyo, F. Guirakhoo, S. Fenner, Z.-X. Zhang, T. P. Monath, and T. J. Chambers, J. Virol. 75:934-942, 2001). The E279 mutation is located in a beta-sheet in the hinge region of the E protein that is responsible for a pH-dependent conformational change during virus penetration from the endosome into the cytoplasm of the infected cell. In independent transfection-passage studies with FRhL or Vero cells, mutations appeared most frequently in hinge 4 (bounded by amino acids E266 to E284), reflecting genomic instability in this functionally important region. The E279 reversion caused a significant increase in neurovirulence as determined by the 50% lethal dose and survival distribution in suckling mice and by histopathology in rhesus monkeys. Based on sensitivity and comparability of results with those for monkeys, the suckling mouse is an appropriate host for safety testing of flavivirus vaccine candidates for neurotropism. After intracerebral inoculation, the E279 Lys virus was restricted with respect to extraneural replication in monkeys, as viremia and antibody levels (markers of viscerotropism) were significantly reduced compared to those for the E279 Met virus. These results are consistent with the observation that empirically derived vaccines developed by mouse brain passage of dengue and YF viruses have increased neurovirulence for mice but reduced viscerotropism for humans.

FIG. 1.

Plaque size variation. (A) ChimeriVax-JE FRhL₃ (large plaque); (B) ChimeriVax-JE FRhL₅ (small plaque). Plaques were stained using rabbit anti-JE virus antiserum followed by anti-rabbit immunoglobulin G-horseradish peroxidase.

FIG. 2.

Survival distributions for YF-VAX and ChimeriVax-JE constructs with and without a mutation at E279 (M→K). Four-day-old suckling mice were inoculated by the i.c. route with approximately 0.7 log₁₀ PFU (A), approximately 1.7 log₁₀ PFU (B), and approximately 2.7 log₁₀ PFU (C).

FIG. 3.

Regression analysis of mortality versus virus dose, showing similar slopes and parallel lines for viruses with (FRhL₅) and without (FRhL₃) the Met→Lys reversion, allowing statistical comparison. The FRhL₅ virus was 18.52 times more potent (virulent) than FRhL₃ (P < 0.0001).

FIG. 4.

Independent RNA transfection and passage series of ChimeriVax-JE virus in FRhL and Vero cells, showing emergence of mutations in the prM-E genes by passage level.

FIG. 5.

Three-dimensional model of the JE virus envelope glycoprotein ectodomain showing locations of mutations in the hinge region occurring with adaptation in FRhL or Vero cells (see Fig. 3). The sequence of the JE virus envelope glycoprotein (strain JaOArS982) (32) was aligned to one of the TBE virus structural template (31) as an input for automated homology modeling building by the method of SegMod (segment match modeling) using LOOK software (Molecular Application Group, Palo Alto, Calif.). The three domains are colored red (domain I), yellow (domain II), and blue (domain III).