12498 ARROYO ET AL. J. VIROL.
TABLE 1. Switch oligonucleotides used for site mutagenesis
E protein position and residue Primerb Marker site
107L3F 5 -CAACGGCTGCGGATTTTTTGGCAAAGGATCCATTGACACATGCGCC-3 BamHI
138E3K 5 -GAAAGAGAATATTAAGTACAAAGTGGCCATTTTTGTCC-3 SspI
*176V 5 -GCCCTCGAGCGGCCGATTCAGCATCACTCCTGCTGCGCCTTCAGTCACAC-3
*176Y 5 -GCCCTCGAGCGGCCGATTCAGCATCAC-3
280K3M 5 -GCAACACTGTCATGTTAACGTCGGGTCATTTG-3 HpaI
316A3V 5 -CTTGGGACTCCCGTGGACACCGGTCACGGCAC-3 AgeI
440K3R 5 -GGGGTGTTCACTAGTGGTTGGGCGGGCTGTCCATCAAGTG-3 SpeI
Primers indicated with an asterisk are cloning primers used in fragment subcloning. One incorporates a change to valine as indicated.
Primers for site-directed mutagenesis to create YF/WN chimeric viruses. Nucleotide changes that switch to a new amino acid are indicated in bold. Silent restriction
(marker) sites introduced are underlined.
generated DNA fragments that were gel puriﬁed and ligated in vitro to produce Neutralizing antibody titers were determined by a constant virus-serum dilu-
a full-length chimeric cDNA. The cDNA was linearized with XhoI to facilitate in tion 50% plaque reduction neutralization assay test (PRNT50) in Vero cells, as
vitro transcription by SP6 polymerase (Epicentre, Madison, Wis.). previously described (24). An equal volume (0.1 ml) of virus suspension contain-
Point mutations were introduced into various E gene codons to produce ing 700 PFU/ml and serial twofold dilutions of heat-inactivated serum were
variants of the original chimera coding for wild-type WN virus prME genes incubated overnight at 4°C, and the serum-virus mixture was inoculated onto
(Transformer site-directed mutagenesis kit; Clontech, Palo Alto, Calif.). Table 1 Vero cell monolayers grown in 12-well plates. An overlay of methylcellulose in
shows the mutation target sites and the oligonucleotide sequences used to create minimal essential medium was added before incubation of the cultures at 37°C
all of the YF/WN chimeras. Site mutations were conﬁrmed by sequencing of the for 3 to 4 days prior to ﬁxation and crystal violet staining for plaque count de-
envelope proteins (prME region) of the resulting viruses. Virus cDNA templates termination. The endpoint neutralization titer was the highest dilution of serum
for sequencing originated from RNA extraction of virus containing infected Vero that reduced plaques by 50% compared to a mouse hyperimmune serum control.
cell supernatants (Trizol LS; Invitrogen, Carlsbad, Calif.) followed by RT-PCR Nonhuman primate studies. Neurovirulence tests in rhesus macaques were
Downloaded from jvi.asm.org by on April 13, 2007
(XL-PCR kit; Applied Biosystems) and sequencing with a CEQ 2000XL nucleic performed according to World Health Organization (WHO) guidelines for test-
acid sequencer (Beckman-Coulter, Fullerton, Calif.). ing YF vaccine (36) and as described previously for safety tests of ChimeriVax-JE
Viruses and cell lines. The wild-type WN virus used in animal challenge vaccine (24). Animals were inoculated with speciﬁc virus candidates by inocula-
studies is the NY99 strain (NY99-35262-11 ﬂamingo isolate, a homolog of the tion of the frontal lobe of the brain (see Table 7). Blood samples were obtained
virus used to build chimeras) obtained from the Centers for Disease Control and daily for the ﬁrst 10 days following inoculation, and serum viremia was measured
Prevention, Fort Collins, Colo. (CDC stock designation B82332W) with two by plaque assay on Vero cells. Animals were observed daily for clinical signs of
additional passages in Vero E6 cells to produce a master virus bank. YF 17D is encephalitis and associated symptoms such as fever or tremors. Animals were
a commercial vaccine (YF-VAX; Aventis Pasteur, Swiftwater, Pa.) used after euthanized 30 days after infection, and the brain and spinal cord tissues were
reconstitution of the lyophilized product or after one passage (P1) in Vero E6 removed for histopathology. Slides were prepared from tissues of the frontal and
(American Type Culture Collection [ATCC] origin; Acambis, Inc., cell bank, temporal cortex, basal ganglia/thalamus (two levels), midbrain, pons, cerebellum
Cambridge, Mass.). Chimeric YF/WN (i.e., ChimeriVax-WN) viruses were pre- (two levels of the nuclei and cortex), medulla oblongata, and six levels of each of
pared by RNA transfection (P1 virus) of Vero E6 cells (ATCC origin, CIDVR cervical and lumbar enlargements of the spinal cord. Sections were stained with
University of Massachusetts Medical Center cell bank, Worcester, Mass.). Re- gallocyanine. Histological lesions were analyzed and scored for pathology rela-
search master seeds (RMS) were prepared by additional ampliﬁcations (either tive to that of the YF 17D according to the criteria for evaluation of neuroviru-
passage 2 or 3 at a 0.001 multiplicity of infection [MOI]) in Vero E6 cells. Vero lence in rhesus monkeys proposed by the current WHO requirements. Mean
E6 cells were maintained in minimal essential medium (Invitrogen) containing lesion scores for individual monkeys were calculated for “target” (substantia
10% heat-inactivated fetal bovine serum (HI-FBS) (HyClone, Logan, Utah). nigra) and “discriminator” (basal ganglia/thalamus and the spinal cord) areas
Preparation of pre-master seeds (PMS) for manufacture of the vaccine was individually and for the target and discriminator areas combined.
initiated by RNA transfection of serum-free Vero (SF-Vero) cells obtained from A second neurovirulence test was performed with cynomolgus monkeys and
a cell bank that had been manufactured and controlled to meet current Food and inoculation with the YF/WNFVR vaccine candidate (ChimeriVax-WN02) produc-
Drug Administration guidelines for cell culture vaccines. (The cells were ob- tion virus seed (P4). The study was conducted according to good laboratory
tained from an ATCC strain predating 1980, and the cell bank was made by practices (GLP) standards (14a). Eleven monkeys were inoculated i.c. with YF/
Baxter/Immuno, Orth, Austria.) Progeny virus from the transfection step was WNFVR production virus seed (P4), 11 positive control monkeys received YF-
ampliﬁed by a single passage in the same SF-Vero cell line to produce P2, which VAX, and 5 negative control monkeys received diluent. The monkeys were eval-
was designated the PMS for subsequent manufacture of clinical-grade vaccine. uated for changes in clinical signs (twice daily), body weight (weekly), and food con-
The SF-Vero cell line is propagated and maintained in a serum-free, animal sumption (daily). Clinical signs were assigned scores according to a clinical scoring
protein-free medium formulation, VT-Media (Baxter/Immuno). Viruses for an- system, based on the WHO requirements for YF vaccine (36). Blood samples
imal experiments were diluted in M199 with HEPES buffer (Invitrogen) and 20% were collected preinoculation on day 1 and on days 3, 5, 7, 15, and 31 for clinical
HI-FBS (HyClone) unless otherwise indicated. Plaque assays to verify the titer of pathology analysis (serum chemistry and hematology parameters). Additional blood
virus inoculi were performed in a Vero cell substrate as previously described (24). samples were collected preinoculation on day 1 and on days 2 to 11 for viremia
Mouse studies. Protocols for mouse experiments were approved by the Insti- analysis, and on days 1 (predose) and 31 for antiviral antibody titer analyses.
tutional Animal Care and Use Committees at both University of Massachusetts To determine immunogenicity, rhesus monkeys were inoculated by the sub-
Medical Center (Worcester, Mass.) and Acambis, Inc. (Cambridge, Mass.). Re- cutaneous (s.c.) route with a single 0.5-ml dose containing 4 log10 PFU of
search was conducted in compliance with the Animal Welfare Act and other chimeric vaccine. Control animals received undiluted YF-VAX containing 4.49
federal statutes and regulations relating to animals and experiments involving log10 PFU in a 0.25-ml volume. Each vaccine dose was back titrated following
animals and adhered to principles set forth in the Guide for the Care and Use of immunization. Serum viremia was measured daily by plaque assay through day 10
Laboratory Animals (27a). Female ICR mice (Taconic, Germantown, N.Y., or after vaccination. Neutralizing antibody levels were measured by PRNT50 on days
Harlan Sprague-Dawley, Indianapolis, Ind.) were inoculated intraperitoneally (i.p.) 14, 30, and 63 after vaccination. Animals were challenged 64 days after vaccination
with 100 to 200 l of wild-type WN virus NY99 for neuroinvasion tests or post- by i.c. inoculation of 125 l containing 2.4 105 PFU of wild-type WN NY99
vaccination challenge experiments (titers of inoculated viruses are indicated in the suspended in M199 with HEPES buffer (Invitrogen) and 10% sorbitol (Sigma).
Results section and in the tables presented). ICR strain adult (3 to 4 weeks of age) Monkeys were observed for viremia, clinical illness, and antibody response;se-
and suckling (2 and 8 days of age) mice were inoculated intracerebrally (i.c.) on verely ill animals were euthanized. The i.c. challenge model closely followed the
the right side of the brain as previously described (24) and using a 20- l volume of model established during the development of ChimeriVax-JE vaccine (24, 26).
YF 17D or chimeric YF/WN constructs for neurovirulence testing (titers of inocu- Genetic stability (in vivo and in vitro passage) and sequencing. The chimeric
lated viruses are indicated in the Results section and in the tables presented). YF/WN virus containing unmodiﬁed, wild-type WN virus prME sequence (des-
Mice were observed daily for 21 days following inoculation to determine ignated ChimeriVax-WN01) was passed six times in Vero E6 cells followed by
survival ratio and average survival time (AST) after virus challenge. six passages in suckling mice by the i.c. route. The chimeric YF/WN virus
VOL. 78, 2004 ChimeriVax-WEST NILE VACCINE PRECLINICAL EVALUATION 12499
TABLE 2. Neuroinvasiveness of ChimeriVax-WN01 relative to The strategy for this mutagenesis approach was to design a safe
YF 17D based on dose response in ICR micea attenuated WN vaccine; this strategy was ﬁrst discussed in an
Back titration dose % Mortality earlier publication (4). Brieﬂy, the selection of speciﬁc amino
Test article i.p.
(log10 PFU) (no. dead/no. tested)d acid residues for mutagenesis was deﬁned by previous studies
ChimeriVax-WN01 (P2)b 0.89 0 (0/5) of the attenuating mutations in a vaccine strain of JE virus
2.23 0 (0/5) (SA14-14-2) (3). Since the wild-type JE and WN viral E gene
3.24 0 (0/5) sequences are identical at the residues implicated in attenua-
4.06 0 (0/5)
5.45 0 (0/5) tion of JE (SA14-14-2) vaccine, with one exception at residue
6.51 0 (0/5) 176, we postulated that introduction of mutations at the ma-
jority of these sites into wild-type WN virus prME genes would
YF17D (ATCC) 2.78 0 (0/3)
4.48 0 (0/3)
result in a similar attenuation of the WN phenotype. Amino
acid residues mapping to the wild-type WN envelope (E) gene
Negative control NAc 0 (0/3) positions 107, 138, 176, and 280 were all mutated in a single
Harlan-Sprague, ICR strain (3 to 4-week-old female mice). construct to encode amino acid residues F, K, V, and M, respec-
P2 indicates a second-generation passage virus on Vero cells. West Nile virus tively. The new chimeric virus was identiﬁed as YF/WNFKVM.
strains are typically neuroinvasive after i.p. inoculation as shown by others (5). Chimeras were constructed in which each amino acid residue in
NA, not applicable.
AST was not determined. the FKVM group was individually mutated to produce single-site
mutants and to assess their individual roles in neurovirulence
(Table 4). The dissection of the FKVM group into single site
containing three mutations introduced by site-directed mutagenesis (designated
ChimeriVax-WN02) at the P2 level (PMS) and P3 level (RMS) were passed 12 mutations identiﬁed only residue 107 as reducing virulence
and 10 times, respectively, in serum-free, protein-free SF-Vero cell substrate. All signiﬁcantly (0% mortality in three mouse neurovirulence tests
in vitro virus passages were performed with an initial MOI of 0.01 PFU/cell presented). Residue 280 followed with 0% mortality after a 105
Downloaded from jvi.asm.org by on April 13, 2007
followed by harvest of the virus on the third day after infection. Passages in vivo viral dose; however, inconsistency of this attenuated phenotype
were performed by initial i.c. inoculation of 105 PFU; brain tissue from ICR mice
(Taconic) was harvested 3 days after inoculation and homogenized, and the
(i.e., mortality ratios of 40 to 89%) was observed in the lower-
clariﬁed homogenate was used for passage to a new group of mice. Virus titers viral-dose groups tested. A mutation at residue 138 resulted in
at each passage were determined by plaque assay. Neurovirulence of the pas- minimal reduction of virulence ( 60% mortality), while a mu-
saged viruses was determined by i.c. inoculation of adult or suckling mice (see tation at residue 176 showed no impact. The neurovirulence of
Tables 13 and 14). Sequencing of viral RNA was performed with Superscript II
the multisite YF/WNFKVM construct resulted in 0 to 20% mor-
reverse transcriptase and XL-PCR; products were puriﬁed by QIAGEN gel
extraction (QIAGEN, Valencia, Calif.). Sequencing reactions were prepared and tality. In later studies, amino acid residues 316 and 440 were
analyzed using the standard Beckman CEQ 2000XL protocol and equipment mutated to V and R, respectively, based on previous data in-
(Beckman-Coulter). For virus passages, at least two independent sequencing dicating mutations in the E protein which mapped to these
reactions were executed per RT-PCR product strand sequenced; sense and regions thought to function in the biology of the E protein
antisense strands were sequenced each time. Mutation acceptance criteria
needed a positive identity in at least three of four sequencing reactions analyzed;
third domain (1, 32). Changes in neurovirulence of these
in addition, two independent operators read sequence chromatographs. mutants with respect to parental ChimeriVax-WN01 were
evaluated in the mouse model as for the previous groups
above (Table 5). A single mutation at residue 316 resulted in a
greater attenuation ( 30% mortality) than residue 440 but not
Virulence phenotype of chimeric YF/WN containing wild-
type WN prME genes relative to YF 17D (YF-VAX). The
initial WN virus chimera encoded the envelope and premem-
TABLE 3. Neurovirulence of ChimeriVax-WN01 relative to
brane protein genes of the WN NY99 wild-type strain (desig- YF 17D based on dose response in ICR micea
nated ChimeriVax-WN01). This chimeric virus did not cause
encephalitis after i.p. inoculation at doses of 106 PFU in 3- to Back % Mortality
Test article i.c. titration dose (no. dead/no.
4-week-old adult ICR mice (Table 2). Encephalitis was as- (days)
(log10 PFU)b tested)
sessed by daily observations for illness, paralysis, and death.
ChimeriVax-WN01 (P2) 2 0 (0/5)
ChimeriVax-WN01 resembles YF 17D vaccine (33) in being 0.30 0 (0/5)
nonneuroinvasive in adult mice. In contrast, the WN NY99 0.89 20 (1/5) 11
wild-type virus was lethal for mice when inoculated by the i.p. 2.23 0 (0/5)
route with as few as 1 to 4 PFU (5; unpublished results). 3.24 20 (1/5) 10
4.06 60 (3/5) 9
ChimeriVax-WN01 retained the ability to cause lethal en- 5.45 20 (1/5) 9
cephalitis after i.c. inoculation, a property consistent with
that of YF 17D virus (10). We estimated the i.c. 50% lethal YF17D (ATCC) 0 20 (1/5) 9
0 60 (3/5) 10.3
dose (LD50) of ChimeriVax-WN01 to be between 103 and 105 0.9 100 (5/5) 9.2
PFU. The neurovirulence phenotype of ChimeriVax-WN01 is 0.98 100 (5/5) 8.2
lower than that of YF 17D virus, for which the i.c. LD50 is 2.78 100 (5/5) 8
between 101 and 102 PFU (Table 3).
Negative control NAc 0 (0/3)
Evaluation of the multisite mutagenesis for attenuation.
Amino acids in the envelope protein previously established Harlan-Sprague, ICR strain (3 to 4-week-old female mice).
Actual dose delivered i.c. assumed to be 20 l for the back titration calcu-
as genetic determinates of virulence for ChimeriVax-JE lations shown.
were changed to reduce the virulence of YF/WN chimeras. c
NA, not applicable.
12500 ARROYO ET AL. J. VIROL.
TABLE 4. Neurovirulence of ChimeriVax-WN01 (YF/WN) TABLE 5. Neurovirulence of ChimeriVax-WN01 site-directed
site-directed mutagenesis variants at E protein residues 1073F, mutagenesis variants at E protein residues 1073F,
1383K, 1763V, 2803M, tested by i.c. inoculation in adult micea 3163V, and 4403R tested in adult micea
Back % Mortality Back % Mortality
Test article Target dose AST Test article i.c. AST
titration dose (no. dead/no. titration dose (no. dead/no.
(Vero passage)b (log10 PFU) (days) (Vero passage) (days)
(log10 PFU) tested) (log10 PFU) tested)
ChimeriVax-WN01 (P3) 4 4.87 100 (5/5) 8.60 ChimeriVax-WN01 (P3) 4.11 83 (10/12) 9.20
5 6.09 60 (3/5) 9 4.74 60 (3/5) 10.33
4.83 100 (8/8) 10.63
YF/WN107F (P2) 4 4.22 0 (0/5)
4 4.42 0 (0/8) YF/WN316V (P3) 4.09 25 (3/12) 12.33
5 4.99 0 (0/5) 4.67 38 (3/8) 10.67
4.57 38 (9/24) 11.22
YF/WN138K (P3) 4 4.26 60 (3/5) 10.33
4 4.41 63 (5/8) 11.40 YF/WN440R (P3) 4.17 83 (10/12) 9.22
5 5.48 60 (3/5) 9.33 4.60 38 (3/8) 10.33
4.35 56 (14/25) 11.21
YF/WN176V (P3) 4 4.42 80 (4/5) 12.50
5 5.54 80 (4/5) 11 YF/WN316V440R (P3) 3.90 17 (2/12) 16.5
4.12 40 (2/5) 13
YF/WN280M (P3) 4 4.14 40 (2/5) 9
3.71 36 (9/25) 12
4 4.55 89 (7/8) 11.86
5 5.14 0 (0/5)
YF/WN107F316V440R (P4) 3.72 0 (0/12)
YF/107F138K280M (P2) 4 3.70 0 (0/5) 5.54 0 (0/12)
5 4.81 0 (0/5) a
Taconic, ICR strain, 3 to 4-week-old female mice. Results of independent
experiments are shown.
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YF/107F138K176V280M (P3) 4 4.13 0 (0/5)
5 5.10 20 (1/5) 7
YF-VAX 3 2.77 100 (5/5) 9 lence of the good manufacturing practice (GMP) manufac-
WN NY99 4 3.90 100 (5/5) 5
tured ChimeriVax-WN02 production virus seed (P4) and a
vaccine lot (P5) prepared for clinical trials. Four litters (32
Taconic, ICR strain (3 to 4-week-old female mice). mice) of 8-day-old suckling mice were inoculated by the i.c.
P2 and P3 indicate second and third generation virus passage on Vero cells,
respectively. route with 20 l containing 103, 104, or 105 PFU of either
production virus seed (P4) or vaccine (P5) virus. Control ani-
mals of the same age received either 103or 105 PFU of YF-
as signiﬁcant as residue 107. The single mutation at residue 440
VAX. Negative controls were inoculated with diluent. The
resulted in a greater level of attenuation over those at residues
results are shown in Table 6. There were no differences across
138 and 176, but only in two of the three independent tests
dose groups in the mortality ratios, and therefore data from
performed (i.e., 40% mortality observed with a mutation at
dose groups for each test article were combined for statistical
residue 440). In summary, neurovirulence of the YF/WN chi-
analysis. There was no difference in the mortality ratio of
meras in which modiﬁed amino acids were inserted in the E
animals infected with P4 or P5. Both the production virus seed
protein at residues 107, 316, and 440 were the most important
(P4) and the vaccine (P5) were highly attenuated compared to
contributors to neurovirulence. Based on this information, a
YF-VAX. The neurovirulence proﬁle of the WN vaccine is
multisite YF/WN107F316V440R construct was selected as our
therefore similar to that of the ChimeriVax-JE vaccine, which
vaccine candidate (ChimeriVax-WN02).
is currently in phase II clinical trials (25).
Neurovirulence studies in mice and in rhesus and cynomolgus
In a pilot monkey neurovirulence study, the ChimeriVax-
macaques. Neurovirulence of viruses with single or multisite mu-
WN01 construct was compared to that of the YF 17D vaccine.
tations in the YF/WN virus E gene was measured in 21-day-old
Rhesus macaques were screened and found negative for ﬂavi-
mice inoculated by the i.c. route with doses between 104 and 105
virus antibodies by hemagglutination-inhibition (HI) test
PFU. This assessment identiﬁed only residues 107 and 280 (Ta-
(kindly performed by Robert Shope, University of Texas Med-
ble 4) and the combination of residues 316 and 440 (Table 5)
ical Branch, Galveston, Tex.). Groups of three young adult
as the dominant attenuating mutations as measured by mor-
tality and AST. The chimera selected as our vaccine candidate
had mutations F, V, and R at residues 107, 316, and 440, respec-
tively, and was avirulent for the adult mouse (Table 5). However, TABLE 6. Comparative neurovirulence of the ChimeriVax-WN02
(YF/WN107F316V440R) vaccine candidate (P5), production virus seed
this virus was neurovirulent for a 2-day-old suckling mouse (data (P4), and YF-VAX in 8-day-old suckling ICR mice (GLP study)
not shown). Because mice become resistant to ﬂavivirus infection
in an age-dependent manner, the suckling mouse is the most % Mortality
(no. dead/no. tested)a
sensitive host for determining subtle differences in neuroviru-
lence. Preliminary studies with ChimeriVax-WN02 virus in suck- Negative control 0 (0/32)
ChimeriVax-WN02 P4 production virus seed 1 (1/96)
ling mice of various ages showed that mice 8 days of age were able ChimeriVax-WN02 P5 vaccine lot 02K01 4 (4/96)
to discriminate differences in neurovirulence, whereas younger YF-VAX 98 (63/64)
mice were too susceptible to differentiate the attenuation phe- a
Statistical signiﬁcance was determined by Fisher’s exact test (two sided).
notype of the ChimeriVax-WN02 vaccine candidate. P 0.0001 for ChimeriVax-WN02 P5 versus YF-VAX, and P 0.3684 for
A GLP study was undertaken to characterize the neuroviru- ChimeriVax-WN02 P4 versus P5.
VOL. 78, 2004 ChimeriVax-WEST NILE VACCINE PRECLINICAL EVALUATION 12501
TABLE 7. Pilot study with rhesus monkeys of neurovirulence of ChimeriVax-WN01 relative to YF-VAX based
on neuropathological evaluation at 30 days post-i.c. inoculations
Back titration dose Individual histopathological score
Test article Monkey Sexa
(log10 PFU) Target area Discriminator area Sum of areas
YF-VAX G211 M 4.40 0.5 0.64 0.59
P417 F 4.40 0 0.43 0.28
N555 F 4.40 1.5 0.66 0.94
Mean SD 0.67 0.76 0.58 0.13 0.60 0.33
ChimeriVax-WN01 N525 M 5.07 1.0 0.58 0.72
D402 M 4.99 0.5 0.48 0.48
C358 F 5.06 0 0.42 0.28
Mean SD 0.50 0.50 0.49 0.08 0.49 0.22
M, male; F, female.
rhesus monkeys were inoculated by the i.c. route with 5 log10 PFU/ml. The number of viremic days did not differ between
PFU of ChimeriVax-WN01 or 4.4 log10 PFU of commercial YF treatment groups (P 0.4067; analysis of variance [AVOVA]).
17D vaccine (YF-VAX) (Table 7). Monkeys inoculated with A higher proportion of monkeys (91%) was viremic on the ﬁrst
the chimera had a mean peak viremia titer of 1.85 0.9 log10 day after inoculation than that seen in the YF-VAX group
PFU/ml with a mean duration of 4.5 days. Monkeys inoculated (27%). On days 2 to 3 after inoculation, the proportion of viremic
Downloaded from jvi.asm.org by on April 13, 2007
with YF-VAX had a similar viremia proﬁle (mean peak vire- monkeys (82%) was the same as for YF-VAX. The mean peak
mia titer of 2.65 0.1 log10 PFU/ml and a mean duration of viremia was 2,097 1,845 PFU/ml. Although the mean peak vire-
4.5 days). Histological scores induced by ChimeriVax-WN01 mia titers for ChimeriVax-WN02 production virus seed (P4)
were lower than those of a higher dose of YF-VAX (Table 7). were higher than that of the reference YF-VAX vaccine (P
Histological lesions in all six monkeys were mildly inﬂamma- 0.0073; ANOVA), individual monkey and group viremia titers
tory, predominantly small perivascular inﬁltrates. The vast ma- for ChimeriVax-WN vaccine remained within acceptable group
jority of them were scored as grade 1 on a scale of 1 to 4. No and individual monkey speciﬁcations, based upon WHO require-
involvement of neurons was seen. The lesions were located ments for YF 17D vaccine (36). The WHO speciﬁcations stip-
mostly in YF vaccine discriminator centers (the basal ganglia/ ulate that no individual monkey will have a viremia exceeding
thalamus areas and both enlargements of the spinal cord). 500 i.c. adult mouse LD50/ml and that no more than 10% of the
Comparison of the two groups of monkeys for the severity and animals will have a viremia exceeding 100 i.c. mouse LD50/ml.
distribution of lesions did not reveal any noticeable difference. We have determined that these limits correspond to 20,000 Vero
On a second neurovirulence study, cynomolgus mon- PFU/0.03 ml and 4,000 PFU/0.03 ml, respectively, in the case
keys were inoculated with YF/WNFVR vaccine candidate of YF-VAX (an LD50 for ChimeriVax-WN02 cannot be deter-
(ChimeriVax-WN02) production virus seed (P4). These ma- mined). The monkey viremias observed following ChimeriVax-
caques were screened and found negative for ﬂavivirus anti- WN02 do not exceed the limits set for YF vaccine.
bodies by HI test (kindly performed by Robert Shope). Eleven There were no abnormalities in hematology or clinical chem-
monkeys were inoculated i.c. with 4.74 log10 PFU of YF/WN- istry values associated with treatment. A complete necropsy
FVR production virus seed (P4), 11 reference control monkeys was performed on day 31, and tissues were prepared for his-
received 5.34 log10 PFU of YF-VAX, and 5 negative control topathology. There were no ChimeriVax-WN02 production
monkeys received diluent. The monkeys were evaluated for seed (P4)-related histopathologic changes in kidney, heart,
changes in clinical signs (twice daily), body weight (weekly), liver, adrenal glands, or spleen.
and food consumption (daily). Clinical signs were assigned
scores according to a clinical scoring system based on the
WHO requirements for YF vaccine (36).
TABLE 8. Summary of CNS histopathologic lesion scores in
YF 17D vaccine virus was detected in the sera of 10 of 11 cynomolgus monkeys inoculated by the i.c. route with ChimeriVax-
monkeys inoculated with YF-VAX. The mean peak viremia WN02 production virus seed (P4), YF-VAX, or negative control
standard deviation (SD) was 357 579 PFU/ml, and the mean
Mean SD lesion scores
number of viremic days was 2.45 1.13. Monkey viremia titers
Treatment group n
were below the 500 and 100 YF-VAX mouse i.c. LD50 values, Target Discriminator Combined
areas areas score
which are the maximum acceptable titers for individual mon-
key and group viremia titers (i.e., present in no more than 10% Negative control 5 0 0 0
of the monkeys), respectively, as established under the WHO ChimeriVax-WN02 11 0.12 0.11 0.13 0.13 0.13 0.09
requirements for YF 17D vaccine. seed (P4)
ChimeriVax-WN vaccine virus was detected in the sera of 10 YF-VAX 11 0.5 0.22 0.54 0.23 0.52 0.2
of 11 monkeys inoculated with ChimeriVax-WN02 vaccine pro- P-valuea 0.000476 0.000357 0.000122
duction seed bank (P4). The duration of viremia was 1 to 5 days a
The Kruskall-Wallis test was used for comparison of the ChimeriVax and
(mean, 2.9 1.38) with peak titers ranging from 180 to 6,400 YF-VAX groups.
12502 ARROYO ET AL. J. VIROL.
TABLE 9. Neutralizing antibody titers (PRNT50) and protective high titers of neutralizing antibodies to the respective virus
activity of ChimeriVax-WN candidate vaccines in with which they were inoculated (data not shown).
adult ICR mice challenged by the i.p. routea
Immunogenicity and efﬁcacy studies in mice and rhe-
Wild-type sus monkeys. The immunogenicity of ChimeriVax-WN01 and
WN NY99 % Survival ChimeriVax-WN02 was evaluated in adult ICR mice inoculated
s.c. dose GMT SD
Vaccine challenge (no. live/
(log10 PFU) (4 wk post- by the s.c. route. Serum neutralizing antibodies were measured
i.p. dose total)
(log10 PFU) by PRNT50 4 weeks after vaccination with a single dose, and titers
ChimeriVax-WN01 3.48 197 93 3 100 (8/8) were expressed as the geometric mean titer (GMT) (Table 9).
In mice, ChimeriVax-WN02 vaccine elicited antibody titers
ChimeriVax-WN02 2.64 20 0 3 40 (4/10) that were approximately 10-fold lower than those elicited by
5.01 37 45 3 100 (9/9)
ChimeriVax-WN01 virus, reﬂecting the greater attenuation of
Negative control NAb 0 3 0 (0/5) this virus. However, when mice were challenged i.p. with 1,000
a LD50 of wild-type WN NY99, mice that had been immunized
Mice were challenged 4 weeks after s.c. vaccination (challenge titer was not
back titrated). with either ChimeriVax-WN01 or -02 were protected in a dose-
NA, not applicable. dependent manner. A vaccine dose of 105 PFU of ChimeriVax-
WN02 protected all animals, whereas a dose of 103 PFU pro-
tected only 40% of the animals.
Histopathology of the brain and spinal cord was performed Young adult rhesus monkeys seronegative for WN neutral-
according to the methods described by Levenbook et al. (20) izing antibodies were vaccinated by the s.c. route with three dif-
and incorporated into the WHO requirements for YF vaccine ferent chimeric vaccines: (i) a chimera containing the E107 (L3
(36). Central nervous system (CNS) lesions were noted in 11 of F) single-site mutation (YF/WNF); (ii) a chimera containing two
11 and 10 of 11 of YF-VAX-treated and ChimeriVax-WN02 mutations at E316 (A3V) and E440 (K3R) (YF/WNVR); and
Downloaded from jvi.asm.org by on April 13, 2007
vaccine-treated monkeys, respectively, and there were no CNS (iii) ChimeriVax-WN02 containing all three mutations.
lesions in the vehicle control monkeys. Inﬂammatory lesions Viremia in the monkeys immunized with the different
induced by both viruses in the meninges and the brain and ChimeriVax-WN viruses following s.c. inoculation was longer
spinal cord matter were minimal to mild (grades 1 or 2) and relative to YF-VAX in some animals, although the levels de-
composed of scanty, mostly perivascular inﬁltrates of mononu- tected at later time points were very low (Table 10). Viremias
clear cells. There was no involvement of neurons in any of the in monkeys receiving the ChimeriVax-WN vaccines ranged
ChimeriVax-WN02- or YF-VAX-treated monkeys. Summary from 1.0 to 2.3 log10 PFU/ml, with a mean duration of 3.5 to 5
data are presented in Table 8. ChimeriVax-WN production days. The mean peak titers of the viremia in monkeys given
virus seed (P4) was signiﬁcantly less neurovirulent (P 0.05) YF-VAX were approximately the same as those receiving the
than the reference article, YF-VAX, in the target, discrimina- WN vaccines. Among the ChimeriVax-WN vaccines, the vire-
tor, and combined mean lesion scores. All monkeys developed mia titers measured suggest an inverse relationship between
TABLE 10. Viremia in rhesus monkeys inoculated by the s.c. route with YF-VAX, ChimeriVax-WN virus constructs with single or
double mutations, and the ChimeriVax-WN02 vaccine candidate
Vaccine and Vaccine dose Viremia (log10 PFU/ml) at day postinoculationa: Mean peak Mean duration
monkey (log10 PFU) 1 2 3 4 5 6 7 8 9 10 titer SD (days)
M017 4.49 0 1.0 2.1 2.9 2.4 0 0 0 0 0 2.4 0.5 3.5
B101 4.49 0 1.6 2.0 1.9 0 0 0 0 0 0
R286 4.49 0 1.8 2.8 2.6 0 0 0 0 0 0
T081 4.49 1.3 1.0 1.5 2.0 0 0 0 0 0 0
N313 4.19 1.6 2.0 1.0 1.3 1.0 0 0 0 0 0 2.2 0.2 5
P367 4.19 0 1.7 1.6 1.8 2.3 1.6 0 0 1.3 0
T087 4.19 2.3 2.3 1.3 1.3 0 0 0 0 0 0
AE81 4.19 2.3 2.1 1.6 1.3 0 0 0 1.0 0 0
R918 4.0 0 2.0 1.5 1.7 0 0 0 0 0 0 1.8 0.2 3.5
N577 4.0 1.0 1.9 1.5 1.0 0 1.0 0 0 0 0
M233 4.0 0 0 1.0 1.0 0 0 0 1.0 1.6 1.8
T757 4.0 0 0 0 1.6 0 0 0 0 0 0
J729 3.92 1.0 0 0 1.0 1.3 1.0 0 1.0 0 1.0 1.4 0.2 4.5
T445 3.92 1.0 1.6 1.5 0 0 1.0 0 0 0 1.0
T086 3.92 1.0 0 1.3 1.3 0 0 0 0 0 0
T491 3.92 0 1.5 0 1.0 0 0 1.0 0 1.0 0
No virus was detected in the assay at day 0 (preinoculation); 1.0 log10 PFU/ml is the assay lower limit.
VOL. 78, 2004 ChimeriVax-WEST NILE VACCINE PRECLINICAL EVALUATION 12503
TABLE 11. Reciprocal neutralizing antibody titers (PRNT50) lenge with WN NY99 (Table 12). It is noteworthy that 50% of
against ChimeriVax-WN virus, rhesus monkeys inoculated by the the animals vaccinated with ChimeriVax-WN developed fever
s.c. route with YF-VAX or ChimeriVax-WN vaccine candidates
after challenge, with an average duration of 5 days postchal-
PRNT50 on day b: lenge, suggesting that they sustained subclinical infections. An
Vaccine and Dose
Postimmunization Postchallenge i.c. challenge with WN virus is extremely aggressive and is the
monkeya (log10 PFU)
only route of challenge tested to induce WN virus disease in
14 30 63 15 31–34
naïve rhesus monkeys. It is likely that virus replication occurs
YF-VAX in brain tissue after i.c. inoculation and before a speciﬁc im-
M017 4.49 320 640 5,120 NAd NA mune response in the brain can be recruited for clearance of
B101 320 640 2,560 1,280 2,560
R286 320 640 640 NA NA the virus. In the case of a human peripherally challenged by a
T081 640 640 2,560 10,240 5,120 mosquito bite, preexisting immunity would rapidly neutralize
the virus and fever is unlikely to occur. However, none of the
GMT 380 640 2,153 3,620 3,620
ChimeriVax-WN-immunized animals developed detectable
viremia after challenge, none developed signs of illness (aside
YF/WN107F from fever), and none died. Vaccinated animals showed an
N313 4.19 160 640 640 2,560 5,120
P367 40 640 640 5,120 2,560
increase in antibody levels postchallenge (Table 11), suggesting
T087 40 640 640 2,560 1,280 that viral replication and antigenic stimulation occurred with-
AE81 40 640 160 10,240 20,480 out associated illness.
Postchallenge viremias ( 102 to 103 PFU/ml) were detected
GMT 57 640 453 4,305 4,305
in the control monkeys that had previously been immunized
with YF-VAX (Table 12). Two out of four monkeys vaccinated
YF/WN316V440 with YF-VAX (M017 and R286) developed a high fever and
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R918 4.0 40 320 640 1,280 2,560
N577 40 160–320c 320 1,280 2,560 signs of encephalitis: muscle tremors, anorexia, and spasticity.
M233 40 160–320c 320 640 1,280 These two animals were euthanized between days 9 and 11
T757 40 40 640 1,280 5,120 after challenge. The other two YF-VAX-vaccinated animals
GMT 40 135 453 1,076 2,560 developed fever and survived i.c. challenge with WN NY99
strain without any clinical symptoms; this ﬁnding is attributed
to cross-protection across the two ﬂaviviruses.
J729 3.92 40 320 80 5,120 5,120
Two monkeys without any prior vaccination were also chal-
T445 80 640 160 640 5,120
T086 160 320–640c 640 1,280 5,120
T491 80 320 160 2,560 5,120
TABLE 12. Viremia and clinical outcome in rhesus monkeys
GMT 80 381 190 1,280 5,120
immunized with ChimeriVax-WN or YF-VAX and
challenged 63 days later by the i.c. route with
For GMT, if an endpoint was not reached, the assay limit titer was used in the 5.38 log10 PFU of wild-type WN NY99 virus
calculation (e.g., 640 taken as 640 and 40 was taken as 40).
PRNT50 was calculated after subtraction of the PRNT from day 0 serum Viremia by day post-i.c. No. of monkeys with
samples. Vaccine and challenge (log10 PFU/ml) outcome/total (%)
PRNT50 calculation fell between the titers shown. The lower titer was used monkey
for the GMT calculation. 1 2 3 4 5 Illness Death
NA, not applicable.
K396 2.0 3.1 2.6 2.4 0 2/2 (100) 2/2 (100)
P500 2.0 3.1 2.6 2.2 1.0
the number of attenuating mutations in the chimera and the YF-VAX
peak titer of viremia (Table 10), but small sample size pre- M017 2.5 2.6 1.7 1.3 0 2/4 (50) 2/4 (50)
cludes deﬁnitive characterization of these differences. B101 2.6 2.5 1.7 0 0
The immunogenicities of vaccine candidates with one, two, R286 3.3 3.3 1.7 0 0
T081 2.4 3.0 2.4 0.5 0
or three attenuating mutations were similar (Table 11). Neu-
tralizing antibody titers ranged from 40 to 640 depending YF/WN107F
upon the vaccine. There were no signiﬁcant differences in N313 0 0 0 0 0 0/4 (0) 0/4 (0)
P367 0 0 0 0 0
neutralizing antibody response between treatment groups (Ta- T087 0 0 0 0 0
ble 11). High titers of neutralizing antibodies ( 100 PRNT50) AE81 0 0 0 0 0
were present 30 and 63 days after vaccination. The observation
that monkeys developed neutralizing antibodies by day 14 in- YF/WN316V440R
R918 0 0 0 0 0 0/4 (0) 0/4 (0)
dicates that ChimeriVax-WN02 elicits rapid onset of protective N577 0 0 0 0 0
immunity. M233 0 0 0 0 0
Rhesus monkeys vaccinated with YF-VAX developed neu- T757 0 0 0 0 0
tralizing antibodies against YF 17D with GMTs of 380 on day ChimeriVax-WN02
14 postvaccination and 2,153 by day 63, which was 1 day before J729 0 0 0 0 0 0/4 (0) 0/4 (0)
challenge with the virulent WN NY99 virus. T445 0 0 0 0 0
Monkeys immunized with ChimeriVax-WN single, double or T086 0 0 0 0 0
T491 0 0 0 0 0
triple mutants were uniformly protected against lethal i.c. chal-
12504 ARROYO ET AL. J. VIROL.
TABLE 13. Neurovirulence of YF/WNFVR RMS (P4 and P11)a relative to YF/WNFVR PMS (Table 13). During all serial pas-
and PMS (P2 and P10)a in 2-day-old ICR strain sages of the virus in Vero cells or brain tissue, no reversions
mice relative to YF 17Db
were detected at target E protein amino acid residues 107F,
% Mortality AST
316V, or 440R, the attenuation markers for the vaccine
Virus i.c. Mutation titration dose candidate. Additionally, during scale-up manufacturing of the
(no. dead/total) (days)
ChimeriVax-WN02 vaccine, no reversions at these critical res-
ChimeriVax-WN02 idues were detected.
P4 None 1.73 80 (8/10) 14.5
The GMP manufactured ChimeriVax-WN02 production vi-
P11 E336C3S 2.08 60 (6/10) 13.67 rus seed (P4) was used for inoculation of large-scale Vero-SF
cultures grown on microcarrier beads in 100-liter bioreactors.
An additional mutation (L3P) occurred in the vaccine at
P2 None 2.10 60 (6/10) 13 position 66 in the M protein. This mutation was associated with
P10 E313G3R 1.88 70 (7/10) 13.67 production of slightly smaller plaque size. The vaccine lot (P5)
contained equal ratios of small and large plaques. Virus pop-
YF-VAX NAc 1.90 100 (10/10) 10.6
ulations with and without the M66 mutation were isolated by
Negative control NA 0 (0/10) plaque puriﬁcation and compared to the PMS (no detectable
Viruses were passed in a serum-free (SF-Vero) stationary-cell substrate.
mutations) and the vaccine lot in the suckling mouse model.
Taconic, ICR strain, mice. One litter (10 mice) of 8-day-old mice was inoculated by the i.c.
NA, not applicable. route with 20 l containing 2, 3, or 4 log10 PFU of either large-
plaque or small-plaque virus and observed for 21 days for signs
lenged with WN NY99 virus. The two challenge control ani- of illness and death. For comparative purposes, litters of mice
were inoculated with similar doses of the PMS (P2) and vac-
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mals developed fever between days 5 and 9 postchallenge, with
slight tremors progressing to ataxia and spasticity between days cine lot (P5) viruses. Mice of the same age were also inoculated
10 and 11, and were euthanized between days 10 and 12. with 2 log10 PFU of YF-VAX. Negative controls were inocu-
Genetic stability. In vitro and in vivo substrate-passage stud- lated with diluent (Table 14). There were no differences in
ies with ChimeriVax-WN01 or the YF/WNFVR chimeric vac- mortality ratios across dose groups, and data were combined
cine candidate (ChimeriVax-WN02) were conducted to deter- for analysis. Since the mortality ratio across all treatment
mine genetic stability of the constructs when grown in groups differed (P 0.0001), pairwise comparisons were per-
stationary cell cultures and in brain tissue. After six in vitro formed. The M66 mutation had no effect on mouse neuroviru-
Vero E6 cell passages of the virus followed by six in vivo ICR lence.
adult mouse brain passages of ChimeriVax-WN01, no muta-
tions were selected relative to the wild-type sequence of the DISCUSSION
prM and E genes in the ChimeriVax-WN01 construct nor was The original YF/WN chimeric virus constructed by insertion
there an increase in mouse neurovirulence (data not shown). A of the prME genes from a wild-type WN virus strain was
heterozygous mutation in the E protein at position E336 re- attenuated with respect to the parental YF 17D virus vector,
sulting in a cysteine-to-serine change was identiﬁed following but retained a degree of neurovirulence for adult mice. To
10 in vitro passages of the YF/WNFVR virus in Vero E6 cells. develop a vaccine candidate with a wider margin of safety, we
In a separate study, in vitro passage of YF/WNFVR in SF-Vero selectively introduced mutations in the donor WN virus. Mu-
cells (manufacturing substrate) resulted in selection of a mu- tations introduced into the E protein of the WN donor virus
tation at position E313 that changed the amino acid at that utilized a strategy based on the previous construction of
position from glycine to arginine. Neurovirulence of these pas- ChimeriVax-JE vaccine, which contained donor prME genes
saged viruses for the 2-day-old suckling mice (n 10) inocu- from an attenuated vaccine strain of JE (SA14-14-2 virus) (3,
lated with a nominal 2-log10 PFU dose of viruses including 4, 28). The SA14-14-2 virus contains mutations at six amino
E313 and E336 mutations showed no increase in virulence acid residues (E107, E138, E176, E279, E315, and E439) that
TABLE 14. Neurovirulence of small- and large-plaque viruses isolated from ChimeriVax-WN02 P5 vaccine in
8-day-old ICR mice inoculated i.c.a
P value for b:
Test article Mutation Test article vs Large plaque vs
(no. dead/no. tested)
Negative control YF-VAX small plaque
Sham (negative control) 0 (0/10) 0.0001
PMS (P2) None 13 (4/30) 0.5558 0.0001
Vaccine lot (P5; large and small plaque) E313G3R, M66L3P 23 (7/30) 0.1612 0.0001
Large plaque E313G3R 3 (1/30) 1.000 0.0001 0.3533
Small plaque E313G3R, M66L3P 13 (4/30) 0.5558 0.0001
YF-VAX 100 (10/10) 0.0001
All ChimeriVax-WN02 viruses used in the evaluation contained the site-directed attenuating mutations 107F316V440R. Additional mutations appearing during Vero
cell passage are shown in the table.
Fisher’s exact test (two sided).
VOL. 78, 2004 ChimeriVax-WEST NILE VACCINE PRECLINICAL EVALUATION 12505
play a role in neurovirulence (3). The WN and JE wild-type by three features: (i) loss of neuroinvasion relative to wild-type
gene sequences are conserved at most of these residues (except WN virus; (ii) introduction of three site-directed mutations in
176), suggesting that mutations introduced at these sites in WN two E protein domains, each independently associated with
virus could have the same attenuating effect as they did in the attenuation; and (iii) conservation of the FVR mutations after
case of JE SA14-14-2. As predicted, we found that mutagenesis in vitro passage in manufacturing-related substrates.
of the WN E residues E107, E280 (corresponding to E279 in The safety of ChimeriVax-WN02 was evaluated in a sensitive
JE virus), and E316 (corresponding to E315 in JE virus) caused 8-day-old suckling mouse model and in rhesus monkeys and in
attenuation of the YF/WN virus chimera. Surprisingly, while cynomolgous macaques inoculated by the i.c. route. In all host-
an E138 mutation, E3K, was associated with a marked atten- virus pairings, the chimeric virus proved to be signiﬁcantly less
uation of JE virus (3, 34), a corresponding mutation in the WN neurovirulent than the licensed YF-VAX vaccine. The monkey
gene did not reduce the neurovirulence of the YF/WN virus to safety test was performed as prescribed by current regulations
the expected 0% mortality by the mouse neurovirulence test. applicable to YF vaccines (36) and showed that the vaccine was
Mutation of the E protein at E440 (corresponding to E439 in signiﬁcantly less virulent than YF-VAX. The nonhuman pri-
JE virus) from K3R, a conservative residue change, also re- mate model has been previously used to assess the safety of
duced neurovirulence for mice. A construct with the three other chimeric vaccines against JE and dengue virus (6, 18, 24).
mutations of F, V, and R at positions E107, E316, and E440, After s.c. inoculation of rhesus monkeys, viremias were more
respectively, was designated ChimeriVax-WN02 and was se- erratic and of longer duration in animals immunized with the
lected as the candidate for manufacture of the vaccine for ChimeriVax-WN vaccines than in animals given YF-VAX (Ta-
clinical studies. ChimeriVax-WN02 was not neuroinvasive com- ble 10). The mean peak titer viremia for YF-VAX-vaccinated
pared to WN NY99 virus and had reduced neurovirulence monkeys was 1 log higher than that for the ChimeriVax-
compared to YF 17D vaccine virus. Attenuation of this virus WN02 (triple mutant) vaccine candidate. The longer viremia
was conferred by the mutation at E107, which maps to the observed after immunization with the chimeric viruses suggests
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fusion peptide in the second domain as predicted in the crystal that the viruses replicate in different tissues had different re-
structure of the E protein (1, 12, 32). This amino acid is ticuloendothelial clearance rates from the parental YF 17D
thought to reduce virulence by altering the function of the virus or had different kinetics of immune response. We are
fusion peptide in the natural cycle of the virus replication. The currently studying the sites of replication of ChimeriVax-WN02
additional ChimeriVax-WN02 mutations at positions E316 and and YF-VAX in tissues of cynomolgus macaques and will
E440 map in domain III on the crystal structure of the E report results in a future publication. In addition, future clin-
protein. Residue E316 is thought to be involved in binding of ical trials will assess the magnitude and duration of viremia
tick-borne encephalitis virus to the virus receptor on the cell following ChimeriVax-WN02 and YF-VAX and establish cor-
plasma membrane (1, 32) and thus may play a role in WN virus relations between viremia and adverse events. The low titer of
cell entry. Residue E440 is in the transmembrane region of the the viremia observed in rhesus monkeys after s.c. vaccination
E protein and is believed to be involved in anchoring the E with the chimeric vaccine candidates suggests that ChimeriVax-
protein during its translation in the endoplasmic reticulum; WN02 vaccine has an acceptable phenotype for trials in hu-
hence, a mutation at E440 may be altering the natural associ- mans.
ation of the E protein with prM (2). The K-to-M mutation at The triply mutated virus (ChimeriVax-WN02) vaccine ap-
position E280 that attenuated neurovirulence for mice was not peared to be less immunogenic than the wild-type chimera in
included in the ﬁnal vaccine because it appeared unstable, mice, but performed satisfactorily in nonhuman primates.
similar to the corresponding residue in JE virus E protein ChimeriVax-WN02 vaccine rapidly elicited a neutralizing anti-
sequence (i.e., E279) shown to be unstable during in vitro body response in all rhesus monkeys and provided solid pro-
passage. A reversion to K at position 279 in the JE virus E tection against an aggressive i.c. challenge with 5 log10 PFU of
protein occurred after less than ﬁve passages of the virus in WN NY99 virus.
MRC-5 cells (22). Mutation of residue E176 from Y in the WN A partially protective immune response was observed in two
virus sequence to either V or I, as seen in JE strains, did not of the four rhesus monkeys immunized with YF 17D and
suggest a signiﬁcant change in neurovirulence; therefore, po- subsequently challenged with wild-type WN virus. Previous
sition E176 was not changed in the ﬁnal vaccine candidate observations by others have shown the cross-protective effect
sequence (unpublished results). This observation contrasts to of prior exposure to phylogenetically related ﬂaviviruses and
the previously published results linking a mutation from I to V concluded that potential for protective cross-reactivity is un-
at position E176 in the JE virus envelope protein to neuro- likely to prevent infection and only likely to prevent disease
virulence (3, 28). Other approaches to ﬂavivirus chimeras em- (16). Similarly, we observed that prior YF immunization of
ployed an attenuated dengue virus genome backbone to pro- monkeys did not prevent infection (viremia) after WN virus
duce chimeric dengue virus vaccine candidates against the four challenge, but may have provided an element of protection
major serotypes (15); similarly, a dengue virus has been used to against death. It should be pointed out that the interval be-
deliver the prM and E genes of WN virus, producing an atten- tween YF immunization and challenge was relatively brief and
uated vaccine candidate shown protective in a nonhuman pri- that cross-protection between heterologous ﬂaviviruses often
mate model (30). This dengue/WN virus chimeric construct diminishes over time, probably due to afﬁnity maturation of the
was attenuated by virtue of the chimeric nature and as a result antibody response and waning of T-cell immunity. It is highly
of a 30-nucleotide deletion in the 3 end noncoding region unlikely that YF immunity would provide reliable cross-pro-
(untranslated region) of the virus genome. tection of humans and therefore a speciﬁc, homologous (WN)
Safety of ChimeriVax WN02 (YF/WNFVR) is characterized vaccination strategy must be pursued. This observation is sim-
12506 ARROYO ET AL. J. VIROL.
ilar to a previous report that hamsters vaccinated with YF 17D Rosa and Robert B. Tesh; and from Acambis, Inc., Rich Weltzin,
were somewhat cross-protected against WN virus challenge- Zheng-Xi Zhang, Jian Liu, and Rick Nichols. Thanks go to Denise
Goens for critical review of the manuscript.
induced disease (35). However, in the rhesus model, only those
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porting our mutagenesis approach to ensure vaccine safety. 17. Komar, O., M. B. Robbins, K. Klenk, B. J. Blitvich, N. L. Marlenee, K. L.
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material in 100-liter bioreactors, a mutation at M66 was de- Republic. Emerg. Infect. Dis. 9:1299–1302.
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ACKNOWLEDGMENTS Crise, K. E. Volpe, M. B. Crabtree, J. H. Scherret, R. A. Hall, J. S. Mac-
kenzie, C. B. Cropp, B. Panigrahy, E. Ostlund, B. Schmitt, M. Malkinson, C.
This work was funded by a NIAID R01 grant AI48297 and NIH Banet, J. Weissman, N. Komar, H. M. Savage, W. Stone, T. McNamara, and
grant 5P51-RR00164-41. D. J. Gubler. 1999. Origin of the West Nile virus responsible for an outbreak
We would like to acknowledge contributions from the following: of encephalitis in the northeastern United States. Science 286:2333–2337.
20. Levenbook, I. S., L. J. Pelleu, and B. L. Elisberg. 1987. The monkey safety
independent contributor Inessa Levenbook; from University of Mas-
test for neurovirulence of yellow fever vaccines: the utility of quantitative
sachusetts Medical School, Worcester, Sharone Green, Francis Ennis, clinical evaluation and histological examination. J. Biol. Stand. 15:305–313.
and John Cruz; from the Centers for Disease Control and Prevention, 21. Marﬁn, A. A., and D. J. Gubler. 2001. West Nile encephalitis: an emerging
John Roehrig; from Sierra Division, Charles River Laboratories, Ken disease in the United States. Clin. Infect. Dis. 33:1713–1719.
Draper; from University of Texas, Galveston, Amelia P. Travassos da 22. Monath, T. P., J. Arroyo, I. Levenbook, Z.-X. Zhang, J. Catalan, K. Draper,