Molecular basis for attenuation of neurovirulence of a yellow fever Virus/Japanese encephalitis virus chimera vaccine (ChimeriVax-JE)

Abstract

A yellow fever virus (YFV)/Japanese encephalitis virus (JEV) chimera in which the structural proteins prM and E of YFV 17D are replaced with those of the JEV SA14-14-2 vaccine strain is under evaluation as a candidate vaccine against Japanese encephalitis. The chimera (YFV/JEV SA14-14-2, or ChimeriVax-JE) is less neurovirulent than is YFV 17D vaccine in mouse and nonhuman primate models (F. Guirakhoo et al., Virology 257:363-372, 1999; T. P. Monath et al., Vaccine 17:1869-1882, 1999). Attenuation depends on the presence of the JEV SA14-14-2 E protein, as shown by the high neurovirulence of an analogous YFV/JEV Nakayama chimera derived from the wild JEV Nakayama strain (T. J. Chambers, A. Nestorowicz, P. W. Mason, and C. M. Rice, J. Virol. 73:3095-3101, 1999). Ten amino acid differences exist between the E proteins of ChimeriVax-JE and the YFV/JEV Nakayama virus, four of which are predicted to be neurovirulence determinants based on various sequence comparisons. To identify residues that are involved in attenuation, a series of intratypic YFV/JEV chimeras containing either single or multiple amino acid substitutions were engineered and tested for mouse neurovirulence. Reversions in at least three distinct clusters were required to restore the neurovirulence typical of the YFV/JEV Nakayama virus. Different combinations of cluster-specific reversions could confer neurovirulence; however, residue 138 of the E protein (E(138)) exhibited a dominant effect. No single amino acid reversion produced a phenotype significantly different from that of the ChimeriVax-JE parent. Together with the known genetic stability of the virus during prolonged cell culture and mouse brain passage, these findings support the candidacy of this experimental vaccine as a novel live-attenuated viral vaccine against Japanese encephalitis.

FIG. 1

Ribbon diagram of the E protein structure based on the model of the soluble fragment of TBEV (44). Numbered arrows indicate positions in domain I, II, or III of the E protein of candidate residues involved in the attenuation phenotype of the ChimeriVax-JE virus.

FIG. 2

Graded-dose neurovirulence testing of revertants 11 and 13 by i.c. inoculation of ICR mice. YFV/JEV Nakayama was used as the virulent control. Two independent clones of revertant (Rev.) 11 were used in this experiment. Mortalities at different doses were as follows: 0 logs, revertant 11-1 (1 of 6), revertant 11-2 (1 of 6), revertant 13 (not done), and YFV/JEV Nakayama (2 of 8); 1 log, revertant 11-1 (9 of 14), revertant 11-2 (2 of 6), revertant 13 (6 of 8), and YFV/JEV Nakayama (6 of 8); 2 logs, revertant 11-1 (10 of 14), revertant 11-2 (2 of 6), revertant 13 (7 of 8), and YFV/JEV Nakayama (8 of 8); 3 logs, revertant 11-1 (6 of 14), revertant 11-2 (3 of 6), revertant 13 (8 of 8), and YFV/JEV Nakayama (8 of 8); 4 logs, revertant 11-1 (11 of 14), revertant 11-2 (2 of 6), revertant 13 (8 of 8), and YFV/JEV Nakayama (not done); 5 logs, revertant 11-1 (4 of 12), revertant 11-2 (4 of 5), and revertant 13 and YFV/JEV Nakayama (not done); 6 logs, revertant 11-1 (7 of 8), and revertants 11-2, 13, and YFV/JEV Nakayama (not done). Differences between 11-1 and YFV/JEV Nakayama were significant for doses of 3 logs (P = 0.012), and 5 logs (P = 0.01). Differences between 11-2 and YFV/JEV Nakayama were significant for doses of 2 logs (P = 0.015) and 4 logs (P = 0.015) (Fisher's exact test). Symbols: open diamonds, YFV/JEV Nakayama; open circles, revertant 13; solid circles, revertant 11-1; open squares, revertant 11-2.

FIG. 3

Sequence alignment (CLUSTAL W) of the E proteins of JEV serogroup members in regions surrounding the residues involved in attenuation (indicated in boldface). Residues of the JEV SA14-14-2 strain are shown above the alignments. The hyphen denotes a missing residue in the alignment for JEV. Symbols indicate fully conserved (asterisks), strongly conserved (colons), or weakly conserved (periods) residues. Strains were as follows: JE-Nakayama, JEV Nakayama strain (28); Murray Valley, Murray Valley encephalitis strain (9); Kunjin, Kunjin virus strain (8); WN-RO9750, West Nile virus RO9750 strain (50); St. Louis, St. Louis encephalitis strain (56).