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Journal of Virology, January 2001, p. 934-942, Vol. 75, No. 2
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.2.934-942.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Molecular Basis for Attenuation of Neurovirulence
of a Yellow Fever Virus/Japanese Encephalitis Virus Chimera
Vaccine (ChimeriVax-JE)
Juan
Arroyo,1
Farshad
Guirakhoo,1
Sabine
Fenner,1
Zhen-Xi
Zhang,1
Thomas P.
Monath,1 and
Thomas J.
Chambers2,*
OraVax, Inc., Cambridge, Massachusetts
02139,1 and Department of Molecular
Microbiology and Immunology, St. Louis University Health Sciences
Center, St. Louis, Missouri 631042
Received 11 July 2000/Accepted 11 October 2000
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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 (E138) 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.
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INTRODUCTION |
The genus Flavivirus of
the family Flaviviridae includes many arthropod-transmitted
viral pathogens, including Yellow fever virus (YFV), the
dengue viruses, and members of the Japanese encephalitis virus (JEV) serogroup and the Tick-borne encephalitis
virus (TBEV) complex (33). JEV remains the most
important cause of acute epidemic viral encephalitis worldwide
(57) and has recently expanded its geographic range to
threaten Indonesia and continental Australia (15, 16, 27).
Vaccines available for prevention of JE include both inactivated and
live-attenuated preparations (57). Formalin-inactivated
vaccine produced from mouse brain (JE-Vax) is manufactured in Japan and
licensed for use by travelers and military personnel in the United
States and some European countries. Although it is effective, the
multiple-dose regimen, the relatively high cost, and problems with
reactogenicity (4, 41-43) complicate its use. The
live-attenuated vaccine (JEV SA14-14-2), prepared from primary hamster
kidney cell cultures, is given only in China, as this cell substrate is
not acceptable for worldwide use.
To address the need for a second-generation vaccine against JEV, we
have developed a candidate live-attenuated vaccine that may be
manufactured in cell cultures to modern standards. The vaccine,
ChimeriVax-JE, is a genetically engineered derivative of the yellow
fever vaccine (YFV 17D), in which the genes encoding the structural
proteins premembrane (prM) and envelope (E) of YFV 17D are replaced
with the corresponding genes of the attenuated JEV SA14-14-2 strain
(6). This experimental vaccine causes an active infection
in recipient mice and rhesus monkeys and induces an immunity which is
both rapid in onset and long-lasting after a single dose
(13). Since the E protein contains the critical neutralizing antibody determinants (18, 26), the
protective immune response elicited by vaccination is directed largely
at JEV.
In principle, the attenuated phenotype and safety profile of the
ChimeriVax-JE virus are based on the derivation of all of its genetic
material from proven vaccine strains (YFV 17D and JEV SA14-14-2). YFV
17D vaccine is generally regarded as one of the safest and most
effective live-attenuated viral vaccines ever developed and is licensed
by national control authorities worldwide (34). The
live-attenuated JEV SA14-14-2 vaccine, although less immunogenic than
YFV 17D, has had an excellent safety record during widespread use in
China (19, 57). Since RNA genomes of flaviviruses have
high rates of mutation, it is important to understand the molecular
basis for attenuation of experimental live virus vaccines and to
demonstrate that multiple genetic determinants govern this property.
The amino acid sequences of the prM and E regions of the
nonneurovirulent ChimeriVax-JE virus and a corresponding neurovirulent YFV/JEV Nakayama virus differ by 3 and 10 amino acid residues, respectively (6). To evaluate the importance of the
differences in the E protein for the attenuated phenotype of
ChimeriVax-JE, we constructed a series of intratypic chimeras harboring
either single or multiple amino acid reversions in the E protein.
Neurovirulence was assessed by intracerebral (i.c.) inoculation in
young adult mice.
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MATERIALS AND METHODS |
Cells and viruses.
Vero cells were originally obtained from
Jerry Jennings (U.S. Army Medical Research Institute for Infectious
Diseases, Fort Detrick, Md.) and were maintained in alpha minimal
essential medium (Gibco/BRL, Grand Island, N.Y.) plus 10% fetal bovine
serum (HyClone, Logan, Utah). Construction of the ChimeriVax-JE and
YFV/JEV Nakayama viruses has been described previously
(6).
Plasmid constructions.
Cloning of pBS/JE SA14-14-2, encoding
the E protein region of the primary dog kidney (PDK)-passaged JEV
SA14-14-2 strain (11) from nucleotides 1108 to 2472 (JEV
numbering), was previously described (6). The reversion
K138
E was introduced into this plasmid by subcloning a
110-bp AflIII/NcoI restriction fragment (JEV
nucleotides 1320 to 1430) from pBS/JE Nakayama into pBS/JE SA14-14-2,
resulting in pBS/JE SAF107E138. The two
plasmids pBS/JE SA14-14-2 and pBS/JE SAF107E138
were then used as templates for derivation of plasmids containing
additional reversions by site-directed mutagenesis. Mutagenesis was
performed using the Transformer site-directed mutagenesis kit
(Clontech, Palo Alto, Calif.). Oligonucleotide primers (Table
1) were synthesized at Life Technologies
(Grand Island, N.Y.). These included silent restriction sites to
facilitate screening for the desired mutations. Convenient restriction
sites in some of the mutagenized pBS/JE plasmids were used to create plasmids containing multiple reversions. All plasmids were analyzed by
nucleotide sequencing of the E region for the presence of the engineered mutation and to verify the integrity of nonmutagenized regions. A region encompassing the mutation(s) was then subcloned from
the pBS/JE plasmids into pYFM5.2/JE SA14-14-2 (6), using NheI and EheI restriction sites. In one case, to
generate the single revertant F107
L, a small
NheI/AflIII restriction fragment from pBS/JE
Nakayama was used to replace the corresponding region of pYFM5.2/JE
SA14-14-2.
RNA transcription and transfection.
Full-length cDNA
templates were assembled by in vitro ligation of restriction fragments
derived from pYF5'3'IV/JE SA14-14-2 (6) and the various
mutagenized versions of pYFM5.2/JE SA14-14-2 after digestion with
NheI and BspEI and purification of the
appropriate fragments from agarose gels. The templates were transcribed
using an AmpliScribe SP6 kit in the presence of methylated cap analog (Epicentre, Madison, Wis.). RNA transfection of Vero cells was done in
the presence of Lipofectin (Gibco/BRL), using approximately 250 ng of
RNA transcript as originally described (46). Viruses were
harvested from the cultures approximately 3 days after transfection, and 1/10 of the volume of harvested medium was used for amplification of virus on Vero cells. Amplified viruses (Vero passage 1) were harvested after onset of cytopathic effects, and recovered virus was
quantitated by plaque titration on Vero cells. Viruses were not plaque
purified prior to neurovirulence testing, but nucleotide sequence
analysis was done for each revertant to verify the presence of the
engineered mutation(s) (see below).
Nucleotide sequence analysis.
Total RNA was extracted using
Trizol (Gibco/BRL) either from infected Vero cell monolayers at passage
1 or from homogenates of virus-infected mouse brain (10% [wt/vol] in
phosphate-buffered saline plus 10% fetal calf serum). Sequencing of
the prM-E region for each virus was done using reverse
transcription-PCR-based methods as previously described
(13). Reaction mixtures were analyzed on a model 310 Genetic Analyzer (PE Applied Biosystems, Foster City, Calif.), and DNA
sequences were refined using Sequencher 3.0 software (GeneCodes, Ann
Arbor, Mich.). Amino acid sequence comparisons were performed using
BLAST searches (3), and sequence alignments of related
viruses were performed using CLUSTAL W (55).
Mouse experiments.
All studies involving mice were conducted
in vertebrate animal biosafety level 3 facilities in accordance with
the U.S. Department of Agriculture Animal Welfare Act (9 CFR Parts 1 to
3) as described in the Guide for Care and Use of Laboratory Animals.
Protocols were approved by Institutional Animal Care and Use
committees. Three- to four-week-old outbred mice (ICR) were purchased
from either Harlan Sprague-Dawley (Indianapolis, Ind.) or Taconic
Farms, Inc. (Germantown, N.Y.). Neurovirulence testing was done by i.c. inoculation of anesthetized mice with 0.03 ml of either
phosphate-buffered saline or M199 medium (Gibco/BRL) plus 10% fetal
bovine serum, containing approximately 10,000 PFU of virus. Virus doses
were confirmed by plaque assay of the inocula on Vero cells. Endpoints of the neurovirulence experiments were scored as both mortality ratios
and average survival times (ASTs). Any mice found in a moribund
condition were euthanatized and scored as dead. Statistical significance of mortality differences was measured by proportions of
survivors in test groups versus controls, using Fisher's exact test.
 |
RESULTS |
Design of the intratypic viruses.
The attenuated JEV SA14-14-2
was originally developed in China by prolonged passage of its virulent
JEV SA14 parent in primary hamster cell culture, followed by multiple
passages of plaque-purified virus sequentially using peripheral
inoculation of hamsters and suckling mice, to improve immunogenicity
(summarized in references 1, 37, and 57).
Mutations responsible for the attenuated phenotype of the vaccine
strain were subsequently predicted by comparing its nucleotide sequence
with that of its parent (1, 37, 38, 40). The availability
of the three-dimensional structure of the E protein of TBEV
(44) and the homology-based model for the E protein of JEV
(25) has guided the development of hypotheses about the
roles of amino acid substitutions in the JEV E protein in the
attenuation process. The positions of the residues which differentiate
ChimeriVax-JE from the YFV/JEV Nakayama virus are shown in Fig.
1. Single mutations (at E protein residue
315 [E315] and E439) are found in domain III
and the C-terminal stem-anchor region, respectively. Three mutations
(at E138 and E176/177) map to domain I (central
domain), and the remaining five (at E107, E227,
E264, E279, and E244) map to domain
II (dimerization domain). We designated residues as "cluster
specific," on the basis of their locations within the different
functional regions of the protein. The underlying hypothesis was that
if the full neurovirulence phenotype involves multiple genetic
determinants, distinguishable levels of virulence would correlate with
the extent of reversions in the various clusters. Table
2 shows the list of single and multiple
site revertants of the ChimeriVax-JE virus which were tested in this
study.

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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.
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To determine if a single reversion could alter the neurovirulence
phenotype, each of the residues which differentiate the
E proteins of
the JEV Nakayama and JEV SA14-14-2 strains were
individually reverted
and tested. Residues E
176 and E
177 were
reverted together because of their proximity. Beyond the analysis
of
single revertants, the construction of multiple site revertants
was
complicated due to the large number of possible combinations.
We
therefore adopted a strategy based on the relationship between
passage
history and accumulation of sequential mutations in the
JEV SA14-14-2 E
protein, while also taking into account their
domain-specific
locations. Within domain II, E
107 was regarded
as a
distinct cluster because of its distal location in this fingerlike
region of the protein. E
138, at the base of domain II, was
segregated
because of its probable role as a neurovirulence determinant
for
JEV (
7,
53). The remaining residues in domain II
(E
227, E
264,
and E
279) were
initially reverted together as a single cluster,
based on their general
vicinity within the proximal portion of
the domain. Residue
E
279 was later tested in combination with
residues
E
107 and E
138, as it was suspected to
contribute a dominant
effect in its cluster. Residue E
244
was not included in the construction
of multiple revertant viruses,
because its presence in the parental
JEV SA14 strain suggests that it
is not a virulence determinant
(
13). Residues
E
315 and E
439 were reverted individually due
to
their locations in either domain III or the stem-anchor region,
respectively.
Nucleotide sequence analysis.
The E protein region of each
revertant virus underwent nucleotide sequence analysis at passage level
1 (first amplification of the transfection harvest on Vero cells),
which was the preparation used for mouse neurovirulence testing.
Sequence analysis revealed the presence of the engineered mutations and
only a few additional silent mutations. Revertant 1 (E107)
contained the silent mutation C
T at nucleotide position 294 of the E
gene, which originated from the cDNA fragment subcloned from the pBS/JE
Nakayama plasmid. Every virus containing the reversion at
K138
E also introduced a silent mutation (A
G) at
nucleotide position 372 of the E gene, which originated from the
fragment of JEV Nakayama cDNA used to generate pBS/JE
SAF107E138 (see Materials and Methods). Two
other silent mutations were included during the site-directed
mutagenesis to revert residues E227 and E264
(T
C at nucleotide position 672 and A
C at position 807). Both of
these mutations were detected in the respective revertant viruses.
Mouse neurovirulence testing.
The neurovirulence of single and
multiple site revertants was expressed as the mortality ratio and AST
following i.c. inoculation with virus. The ChimeriVax-JE (YFV/JEV
SA14-14-2) and YFV/JEV Nakayama viruses (both at passage level 1 on
Vero cells) were used as the attenuated and virulent controls,
respectively. Results are shown in Table
3. Revertant viruses were classified into three groups, based on the level of associated mortality. Lethal revertants (mortality ratios of
89% [not significantly different from YFV/JEV Nakayama]) included 13, 16, 17, and 19. Sublethal revertant viruses (mortality ratios between 13 and 38% [significantly different from YFV/JEV Nakayama, but not ChimeriVax-JE]) included 11, 12, 14, and 15. Attenuated revertants included those in which no
mortality occurred, except for revertant 6, for which one of eight mice
succumbed 5 days postinoculation. ASTs for all mice with mortality
endpoints varied from 9 to 13 days, essentially the same as those for
the YFV/JEV Nakayama virus control.
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TABLE 3.
Neurovirulence testing of revertant viruses by i.c.
inoculation of 4-week-old ICR mice with 104 PFU of virus
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Within the group of lethal revertants, revertant 13 contained the
lowest number of substitutions (four). To determine if its
level of
neurovirulence was quantitatively the same as that of
the YFV/JEV
Nakayama virus, graded dose testing was done (Fig.
2). Revertant 11 was also analyzed
because it exhibited a dramatic
difference in mortality from revertant
13, while differing only
by the absence of the E
176/177
cluster. Revertant 13 exhibited
a 50% lethal dose (LD
50)
of less than 10 PFU. For revertant 11,
two independently derived clones
showed variable mortality ratios
over doses between 1 and 100,000 PFU,
making the LD
50 measurement
indeterminant. At the dose of
10,000 PFU, mortality ranged from
35 to 80%. Because of this
variability, revertant 11 was sequenced
using RNA from infected mouse
brains to determine whether new
mutations had occurred during
replication of this virus in vivo.
Analysis of the prM-E region for
three separate samples revealed
no differences from the sequence
determined for the original passage
1 virus. It should be noted that
two different lineages of ICR
mice {Hsd:ICR(CD-1
R) and
Tac:Icr:Ha[ICR]fBR} were used for the initial neurovirulence
testing (Table
3) and the LD
50 determinations (Fig.
2),
respectively.
The former exhibited a mortality ratio of 13% whereas
the latter
exhibited the higher mortality ratios at the 10,000-PFU
dose.
These two random-bred ICR stocks have been sustained using
different
breeding protocols following their common origin as the Fox
Chase
version in 1959. We suspect that genetic differences among these
mice may partly account for the variable neurovirulence properties
of
revertant 11 observed in these experiments.

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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.
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To further investigate the variability in mortality ratios observed
with revertant 11, additional experiments were done with
this
revertant, as well as additional revertants, in ICR mice
of both
lineages. In Ha[ICR]fBR mice, revertants 11, 12, and 13
were compared
at a dose of 4.0 logs using ChimeriVax-JE and YFV/JEV
Nakayama viruses
as controls. Mortality ratios and ASTs were as
follows: revertant 11, seven of eight dead (AST of 10.6 days);
revertant 12, three of nine
dead (AST of 14.7 days); revertant
13, eight of eight dead (AST of 11.1 days); YFV/JEV Nakayama,
eight of eight dead (AST of 9.1 days);
ChimeriVax, zero of eight
dead. In CD-1
R mice, revertants
11, 12, and 15 were compared in an independent
experiment using
ChimeriVax-JE and YFV/JEV Nakayama viruses as
controls. Mortality
ratios were as follows: for revertant 11 at
4.0 logs, one of eight dead
(day 10); for revertant 12 at 4.0
logs, one of eight dead (day 14); for
revertant 12 at 4.7 logs,
zero of seven dead; for revertant 15 at 4.0 logs, one of eight
paralyzed; for revertant 15 at 4.7 logs, zero of
seven dead; for
ChimeriVax-JE at 6.0 logs, zero of eight dead; for
YFV/JEV Nakayama
at 4.0 logs, seven of seven dead (AST of 9.1
days).
Taken together with the results presented in Table
3 and Fig.
2, the
data indicate that a distinct difference in the behavior
of revertant
11 occurs between ICR mice of the two lineages and
that this is not due
to a general increase in the sensitivity
of the Ha[ICR]fBR mice to
these viruses, as neither the ChimeriVax-JE
virus nor another sublethal
revertant differed in their virulence
phenotype between the two mouse
strains. Furthermore, revertant
11 exhibited a variable mortality ratio
across a wide range of
doses, but its phenotype did not resemble that
of the YFV/JEV
Nakayama virus. The neurovirulence properties of
revertant 11
are governed by the strain of mouse employed, and the
revertant
cannot be classified as either completely attenuated or fully
virulent.
 |
DISCUSSION |
We used a previously established genetic system (6)
to test whether the attenuated phenotype of the ChimeriVax-JE virus could be defined in terms of specific amino acid residues of the E
protein. This question is relevant to the use of the virus as a
live-attenuated vaccine against JE. A nominal requirement for such a
vaccine is the presence of multiple mutations which independently contribute to attenuation. In the case of other flaviviruses, this has
commonly been observed among pairs of virulent and attenuated viruses
(14, 24, 40). YFV 17D vaccine, for example, differs from
the parental Asibi strain by a total of 32 amino acid substitutions, 12 of which occur in the E protein and are believed to include virulence
determinants (14). 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.
In general, the severities of the neurovirulence phenotypes correlated
directly with the number of engineered reversions. Single substitutions
(with the exception of revertant 6) produced attenuated viruses.
Further studies are needed to determine whether the 13% mortality rate
for revertant 6 reflects a real neurovirulence difference from the
ChimeriVax-JE virus. Two to three substitutions produced either
attenuated (revertant 10) or sublethal (revertants 11, 12, and 14)
viruses. Four substitutions created a sublethal revertant when two
clusters were included (revertant 15 [E138, E227, E264, E279]) but a lethal
revertant when three clusters were involved (revertant 13 [E107, E138, E176/177]). This
latter combination of four reversions is probably sufficient for
restoration of full neurovirulence, since the LD50 of
revertant 13 (Fig. 2) is similar to that of the YFV/JEV Nakayama virus,
previously determined to be less than 10 PFU (6). More
than four substitutions generated lethal revertants, but only when the
E138 reversion was present (see below). It should therefore
be emphasized that, except for revertant 13, all viruses with four or
fewer substitutions had mortality ratios significantly less than that
of the virulent YFV/JEV Nakayama virus (P
0.01).
The level of neurovirulence was affected not only by the number of
engineered reversions but also by their specific combination. For
example, revertant 18 (E107, E176/177,
E227, E264, E279) was attenuated,
whereas revertant 17 (E138, E176/177,
E227, E264, E279) was fully
neurovirulent. In this case, substitution at the E138
cluster rather than at E107 conferred neurovirulence in the presence of the E176/177 and
E227-E264-E279 reversions. Residue E138 has been identified as a principal virulence
determinant of JEV in the background of the JaOArS982 and NT109 strains
(7, 53). Our data are generally consistent with this
finding. Although a single reversion at E138 was
insufficient to restore neurovirulence, its presence together with any
other reverted cluster (such as in revertants 11, 12, and 15) always
produced viruses with at least sublethal phenotypes. Thus, while
reversion at residue E138 has a dominant effect on
neurovirulence properties of the ChimeriVax-JE virus, it must occur
with multiple other reversions to restore a fully neurovirulent
phenotype. Residue E138 lies in the so-called "hinge"
region at the domain I-II interface of the E protein. Studies with
several flaviviruses have provided data indicating that mutations
within this region modulate virulence phenotypes in mice (5, 7,
12, 17, 29, 30, 53). This is believed to occur through effects
of such mutations on the low-pH-induced dimer-to-trimer structural
transition of the E protein associated with virus entry.
In contrast to those at the E138 cluster, reversions at the
other sites were not absolutely required for reconstitution of lethal
or even sublethal phenotypes. However, reversions at each cluster
contributed to measurable increases in neurovirulence. Reversion at
residue E107 had minimal or sublethal effects in combination with a reversion at a single other cluster (revertants 10 and 11). However, a marked enhancement of virulence occurred in viruses
containing the E107 reversion plus reversions at two or
more additional clusters (when E138 was included). Its
effect in combination with reversions at the
E227-E264-E279 cluster has not been
tested, but this would be expected to result in a sublethal or
attenuated virus. Residue E107 lies within a highly
conserved hairpin motif encompassing amino acids 98 to 111 of domain II (25). This region is believed to contain the fusogenic
peptide, based on studies with Murray Valley encephalitis and dengue 2 viruses (47, 48). Mutations near this region change the
fusion properties of the E protein in cell culture and have been
associated with alterations in neurovirulence (10, 44).
The E176/177 cluster was identified as an independent
virulence determinant by comparison of revertant 13 (E107,
E138, E176/177 [100% mortality]) with
revertant 11 (E107, E138 [13% mortality]; P < 0.005). This conclusion is also supported by
comparison of revertants 15 (E138, E227,
E264, E279) and 17 (E138,
E176/177, E227, E264,
E279), where inclusion of E176/177 increased
mortality from 22 to 100% (P < 0.005). Thus, as for
residues E138 and E107, reversion at the
E176/177 cluster enhanced the virulence of viruses containing reversions at each of the other clusters. Residues E176/177 lie within the central domain of the E protein, a
region enriched in conformational epitopes which are sensitive to low pH. Structural changes associated with the reorganization of the E
protein dimer into the fusion-active trimer are believed to occur
within this domain (2, 52). The region surrounding the
E176/177 cluster has been implicated as a virulence locus on the basis of numerous genetic and functional studies. For YFV, residue E173 is a position where the YFV 17D vaccine
differs from the virulent Asibi, French viscerotropic, and French
neurotropic strains (14, 21). Neuroadaptation of YFV 17D
vaccine to mouse brain is associated with reversion of this residue
(I173
T) (51). In addition, a mutation at
residue E181 in the TBEV affects mouse neurovirulence by
lowering the threshold for low-pH-induced fusion of the E protein
(20). Although we have not demonstrated whether reversions
at both E176 and E177 are required for the
effect of this cluster on neurovirulence, data discussed below suggest
that E176 may be the more important determinant.
Within the E227-E264-E279 cluster,
reversion at E279 appeared to increase neurovirulence,
since revertant 11 (E107, E138) caused 13%
mortality, whereas revertant 14 (E107, E138,
E279) caused 38% mortality, even though this difference
was not statistically significant (P > 0.05). Residue
E279 lies within the hinge region of the E protein and may
affect its properties in a manner similar to that of the
E138 residue. Studies with Murray Valley encephalitis virus
have shown that mutations near this position can impair the
hemagglutination and fusion properties of the E protein and reduce
neuroinvasiveness for mice (29, 31). The full
neurovirulence effect contributed by the
E227-E264-E279 cluster required at
least one additional reversion beyond E279 (compare
revertant 14 with revertant 16 [E107, E138,
E227, E264, E279]; 89% mortality;
P = 0.043). There is some evidence that
E264, rather than E227, accounts for this
effect, since an E227 serine is present in all JEV SA14
strains and their cell culture-passaged derivatives, and the mutation
to proline at this residue occurs instead in the JEV Nakayama strain.
These conclusions about the roles of both the E176/177 and
E227-E264-E279 clusters in
attenuation are also supported by comparison of the sequences of the
parental JEV SA14 strain and its attenuated derivatives (1, 37,
40). The attenuated JEV SA14-5-3 strain (an early progenitor of
JEV SA14-14-2) contains substitutions at E107,
E138, E176, and E279. These
mutations remained stable during sequential primary hamster kidney and
PDK cell passage. However, substitutions at E177 and E264 were unstable after PDK cell passage and reverted back
to the original residues, despite the virus remaining attenuated. Thus,
reversions at these two positions may not independently contribute a
strong effect to neurovirulence. The JEV SA14-2-8 virus, another
attenuated derivative of the SA-14 parent, and not a progenitor of JEV
SA14-14-2, also contains the same mutations at E138 and
E176 as does the JEV SA14-14-2 virus (in addition to three
other substitutions specific to its E protein). Although the data taken
together suggest that E176 and E279 may be the principal virulence determinants of their clusters, the effects of
combinations of substitutions within either cluster on neurovirulence remain unpredictable, and comparisons with E proteins of other attenuated strains may be complicated by the presence of unrelated mutations. Full understanding of the contribution of residues within
the E176/177 and
E227-E264-E279 clusters will
require systematic studies of additional engineered revertants.
With regard to the substitutions in domain III (E315) and
the stem-anchor region (E439), viruses containing single
reversions at these residues were attenuated. However, a revertant
containing both substitutions was not tested in our experimental
system. Residue E315 lies along the distal surface of
domain III, a region which has been previously implicated in the
process of virion attachment to host cells (44). In YFV
and JEV, mutations in the vicinity of E315 are associated
with altered virus tropism and changes in virulence (22, 23, 39,
49). Residue E439 lies within a predicted
alpha-helical segment of the stem-anchor region whose structural
integrity is required for stability of the prM-E heterodimer, based on
studies with TBEV (2). The conservative nature of the
K
R substitution at position E439 in ChimeriVax-JE may
mitigate against any major effect of this mutation on the properties of
the E protein; however, this remains to be tested. In short, we cannot
at present exclude the possibility that a combination of reversions at
residues E315 and E439 could increase the
neurovirulence of ChimeriVax-JE, but determinants in domains I and II
alone are clearly capable of conferring high-level neurovirulence.
Testing of additional combinations of multiple site revertants is
needed to further understand the quantitative contributions of the
various virulence determinants tested here. It should be emphasized
that, in addition to the reversions in the E protein, it remains
possible that effects of mutations in the prM region (where three
conservative substitutions occur between the ChimeriVax-JE and YFV/JEV
Nakayama viruses [6]) could conceivably contribute to
enhanced neurovirulence. We also acknowledge that any conclusions drawn
here about the proposed functional role of the various mutations in the
attenuated phenotype of ChimeriVax-JE remain speculative in the absence
of direct experimental data examining their effects on the structure
and function of the E protein.
Based on the data presented here, we believe that no single amino acid
reversion in the E protein of ChimeriVax-JE to that of JEV Nakayama
could restore a mouse neurovirulent phenotype. This suggests a
relatively stable genetic basis for attenuation of this experimental
vaccine. In fact, ChimeriVax-JE may be safer than the YFV 17D vaccine,
based on clinical experience and collective mouse and primate
neurovirulence data. Vaccination of humans with YFV 17D has caused only
21 known cases of postvaccinal encephalitis, the majority in infants,
despite administration of over 300 million doses (34). No
case of postvaccinal encephalitis has been reported with the use of JEV
SA14-14-2 vaccine (57). Experimentally, neurovirulence
occurs in mice inoculated with YFV 17D at a dose as low as 1.4 log10 PFU, while ChimeriVax-JE causes 0% mortality even at
doses of 6 log10 PFU (6, 13). Moreover,
ChimeriVax-JE is less neurovirulent than YFV 17D in primate
neurovirulence testing (35, 36). Although the results
reported here are promising with regard to the safety profile of
ChimeriVax-JE, they cannot be generalized to the primate system, where
host range differences could influence the virulence profiles of the
revertant viruses. In some cases, however, concordance between mouse
and monkey neurovirulence among flavivirus strains has been observed
elsewhere (32). Appropriate animal testing to establish
the attenuation phenotype in conjunction with verification of stability
of the virus upon serial passage in vitro and in vivo and confirmation
of the vaccine genotype at the final step of manufacturing are minimal
requirements to be met in terms of monitoring for the emergence of a
virulent revertant.
Finally, the results of this study may also be applicable to
understanding the molecular basis of neurovirulence of other closely
related flaviviruses. To address this question, amino acid sequences
surrounding residues E107, E138,
E176, E264, and E279 of the JEV
Nakayama strain (28) were analyzed using the BLAST search
program. Top-scoring E protein sequences were then aligned with CLUSTAL
W (Fig. 3). Viruses with the highest
scores were Murray Valley encephalitis virus (9), Kunjin
virus (8), West Nile virus (WN-RO9750 strain)
(50), and St. Louis encephalitis virus (56),
in that order. The presence of highly conserved amino acid sequences
encompassing positions E107, E138,
E264, and E279 (and a less conserved
E176 region) raises the possibility that mutations at the
equivalent residues or adjacent residues may affect the virulence
phenotypes of the related flaviviruses. Any such mutations would most
likely be constrained by the potential for deleterious effects on
critical functions associated with these various regions of the E
protein, which could reduce viral fitness. Because the E proteins of
the JEV serogroup viruses generally exhibit a high level of amino acid
homology, it is possible that other conserved regions may harbor
additional determinants of neurovirulence not identified here, as other
studies have suggested (39). In any case, identification
of residues in the E protein which govern neurovirulence is a starting
point for further investigations. These might include mutagenesis of
the critical determinants and surrounding residues, in conjunction with
molecular clone technology, to generate additional live-attenuated
virus vaccine candidates in the ChimeriVax background.

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|
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).
|
|
 |
ACKNOWLEDGMENTS |
We thank Brigitte T. Huber and Sue Hurta (Tufts University) for
biosafety level 3 facility support and Chuck Miller, OraVax, Inc., and
Deborah Droll, Saint Louis University, for expert technical assistance.
This work was supported in part by grants from the NIAID (AI-136798
[OraVax] and AI-43512-03 [T.J.C.]), the WHO Global Program for
Vaccines and Immunization (T.J.C.), and the Edward Mallinckrodt, Jr.
Foundation (T.J.C.).
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Microbiology and Immunology, St. Louis University Health
Sciences Center, 1402 S. Grand Blvd., St. Louis, MO 63104. Phone: (314) 577-8447. Fax: (314) 773-3403. E-mail: chambetj{at}slu.edu.
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0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.2.934-942.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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