JOURNAL OF VIROLOGY, May 2001, p. 4040–4047 Vol. 75, No.

9
0022-538X/01/$04.00⫹0 DOI: 10.1128/JVI.75.9.4040–4047.2001

West Nile Virus Recombinant DNA Vaccine Protects Mouse and Horse
from Virus Challenge and Expresses In Vitro a Noninfectious
Recombinant Antigen That Can Be Used in
Enzyme-Linked Immunosorbent Assays
BRENT S. DAVIS,1 GWONG-JEN J. CHANG,1* BRUCE CROPP,1 JOHN T. ROEHRIG,1 DENISE A. MARTIN,1
CARL J. MITCHELL,1 RICHARD BOWEN,2 AND MICHEL L. BUNNING1
Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service,
U.S. Department of Health and Human Services,1 and Colorado State University,2
Fort Collins, Colorado 80522

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Received 15 November 2000/Accepted 29 January 2001

Introduction of West Nile (WN) virus into the United States in 1999 created major human and animal health
concerns. Currently, no human or veterinary vaccine is available to prevent WN viral infection, and mosquito
control is the only practical strategy to combat the spread of disease. Starting with a previously designed
eukaryotic expression vector, we constructed a recombinant plasmid (pCBWN) that expressed the WN virus
prM and E proteins. A single intramuscular injection of pCBWN DNA induced protective immunity, prevent-
ing WN virus infection in mice and horses. Recombinant plasmid-transformed COS-1 cells expressed and
secreted high levels of WN virus prM and E proteins into the culture medium. The medium was treated with
polyethylene glycol to concentrate proteins. The resultant, containing high-titered recombinant WN virus
antigen, proved to be an excellent alternative to the more traditional suckling-mouse brain WN virus antigen
used in the immunoglobulin M (IgM) antibody-capture and indirect IgG enzyme-linked immunosorbent
assays. This recombinant antigen has great potential to become the antigen of choice and will facilitate the
standardization of reagents and implementation of WN virus surveillance in the United States and elsewhere.

Between late August and early September 1999, New York western Asia, Europe, and Australia (18). Clinically, WN fever
City and surrounding areas experienced an outbreak of viral in humans is a self-limited acute febrile illness accompanied by
encephalitis that caused seven deaths with 62 confirmed cases. headache, myalgia, polyarthropathy, rash, and lymphadenopa-
Concurrent with this outbreak, local health officials observed thy (28). Rarely, though, acute hepatitis or pancreatitis has
increased mortalities among birds (especially crows) and been reported, and cases in elderly patients are sometimes
horses. The outbreak was subsequently shown to be caused by complicated by encephalitis or meningitis (7).
West Nile (WN) virus, based on monoclonal antibody (MAb) Currently, no human or veterinary vaccine is available to
mapping and gemonic sequences detected in human, avian, prevent WN virus infection, and mosquito control is the only
and mosquito specimens (4, 17, 22). Virus activity detected practical strategy to combat the spread of disease. Recently, we
during the ensuing winter months (5, 6, 13) indicated that the reported the development of a highly immunogenic recombi-
virus had established itself in North America. In 2000, surveil- nant DNA vaccine for JE virus that induced protective immu-
lance data reported from the northeastern and mid-Atlantic nity in mice following a single intramuscular (i.m.) injection
states confirmed an intensified epizootic-epidemic transmis- (8). COS-1 cells transformed with this plasmid secreted the
sion and a geographic expansion of the virus. Numerous cases premembrane (prM) and envelope (E) proteins in the form of
of infection in birds, mosquitoes, and horses as well as cases in extracellular subviral particles (EPs) into culture medium. We
humans were documented (6). also demonstrated that partially purified EPs not only induced
WN fever is a mosquito-borne flavivirus infection that is a protective immune response in mice, but more importantly
transmitted to vertebrates primarily by various species of Culex could serve as a noninfectious recombinant antigen (NRA) in
mosquitoes. Like other members of the Japanese encephalitis both immunoglobulin M (IgM)-antibody capture (MAC) and
(JE) antigenic complex of viruses, including JE, St. Louis en- indirect IgG enzyme-linked immunosorbent assays (ELISAs)
cephalitis (SLE), and Murray Valley encephalitis viruses, WN (A. R. Hunt and G. J. Chang, submitted for publication).
virus is maintained in a natural cycle between arthropod vec- Because WN virus is closely related to JE virus, a recombinant
tors and birds. The virus was first isolated from a febrile human WN virus plasmid was constructed in this study to investigate
in the West Nile district of Uganda in 1937 (38). It was soon its potential as a source of NRA for diagnosis and as a candi-
recognized as one of the most widely distributed flaviviruses, date DNA vaccine to prevent WN virus infection.
with its geographic range including Africa, the Middle East,
MATERIALS AND METHODS

Cell culture and virus strain. COS-1 cells (American Type Culture Collection,
* Corresponding author. Mailing address: P.O. Box 2087, Fort Col- Manassas, Va.; 1650-CRL) were grown at 37°C with 5% CO2 in Dulbecco’s
lins, CO 80522-2087. Phone: (970) 221-6497. Fax: (970) 221-6476. modified Eagle’s medium (DMEM; Gibco, Grand Island, N.Y.) supplemented
E-mail: gxc7@cdc.gov. with 10% heat-inactivated fetal bovine serum (FBS; Hyclone Laboratories, Inc.,

4040
VOL. 75, 2001 WEST NILE VIRUS DNA VACCINE 4041

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FIG. 1. Map of the WN virus genomic region (top) and oligonucleotides used in RT-PCR to construct the transcription unit for the expression
of WN virus prM and E coding regions (bottom). Potential transmembrane helices of viral proteins are indicated by black boxes.

Logan, Utah), 1 mM sodium pyruvate, 1 mM nonessential amino acids, 17 ml per scribed in a previous publication (8). Electroporated cells were seeded onto
liter of 7.5% NaHCO3, 5 ml per liter of 1 M HEPES (Bio Whittaker, Walkers- 75-cm2 culture flasks or in a 12-well tissue culture dish containing one sterile
ville, Md.), 100 U of penicillin per ml, and 100 ␮g of streptomycin per ml. Vero coverslip per well. All flasks and 12-well plates were kept at 37°C in a 5% CO2
cells were grown under the same conditions used for COS-1 cells. Two WN virus incubator. Forty hours following electroporation, coverslips containing adherent
strains (NY99-6480, a mosquito isolate, and BC787, a bird isolate), isolated from cells were removed from the wells, washed briefly with phosphate-buffered saline
the outbreak in New York in 1999, were used for challenge experiment or (PBS), fixed with acetone for 2 min at room temperature, and allowed to air dry.
mosquito inoculation. The NY99-6480 strain was propagated in the Vero cell The flavivirus E protein-specific MAb 4G2 (17), WN virus mouse hyperimmune
culture. The BC787 strain was passed once in suckling-mouse brain. The NY99- ascitic fluid (HIAF), and normal mouse serum (NMS) at a 1:200 dilution in PBS
6480 strain used for immunological or biochemical studies was gradient purified were used as the primary antibodies to detect protein expression by an indirect
by precipitating in 7% polyethylene glycol 8000 (PEG-8000; Fisher Scientific, immunofluorescence antibody assay (IFA), as described previously (8).
Fair Lawn, N.J.) followed by ultracentrifugation on 30% glycerol–45% potassium Tissue culture medium was harvested 40 and 80 h following electroporation.
tartrate gradients (30). Antigen capture (Ag-capture) ELISA was used to detect secreted WN virus
Construction of plasmid expressing WN virus prM and E proteins. Genomic antigen in the culture medium of transiently transformed COS-1 cells. MAb 4G2
RNA was extracted from 150 ␮l of Vero cell culture medium infected with strain and horseradish peroxidase-conjugated MAb 6B6C-1 were used to capture the
NY99-6480 using a QIAamp viral RNA kit (Qiagen, Santa Clarita, Calif.). WN virus antigens and detect captured antigen, respectively (8, 15, 34).
Extracted RNA, resuspended in 80 ␮l of diethyl pyrocarbonate-treated water
(Sigma, St. Louis, Mo.), was used as a template in a reverse transcriptase-PCR
(RT-PCR) for the amplification of WN virus prM and E genes. Primer sequences
(Fig. 1) were designed based on the published sequence (22). Restriction enzyme
sites for BsmBI and KasI were incorporated at the 5⬘ terminus of the cDNA
amplicon. An in-frame termination codon followed by a NotI restriction site was
introduced at the 3⬘ terminus of the cDNA amplicon. The RT-PCR and molecu-
lar cloning protocols used are essentially identical to those reported previously (8).
The WN virus cDNA amplicon was digested with KasI and NotI enzymes, and
the resulting 998-bp (nucleotides ⫺1470 to 2468) fragment of the cDNA was
inserted into the KasI and NotI sites of a pCBJESS vector to form an interme-
diate plasmid, pCBINT. pCBJESS was derived from the pCBamp plasmid, which
contained the cytomegalovirus early gene promoter and translational control
element and engineered JE virus signal sequence element (8) (Fig. 1). The
cDNA amplicon was subsequently digested with BsmBI and KasI enzymes, and
the remaining 1,003-bp fragment (nt ⫺466 to 1469) was inserted into the KasI
site of pCBINT vector to form pCBWN (Fig. 2). Automated DNA sequencing
was performed on an ABI Prism 377 sequencer (Applied Biosystems/Perkin
Elmer, Foster City, Calif.) according to the manufacturer’s recommended pro-
cedures. The recombinant plasmid which had a correct prM and E sequence was FIG. 2. Map of the recombinant WN virus plasmid pCBWN. The
identified (22). transcription unit contains the human cytomegalovirus early gene pro-
Immunochemical characterization of the recombinant WN virus antigen. moter (CMV), JE virus signal sequence, WN virus prM and E gene
COS-1 cells were electroporated with plasmid pCBWN using the protocol de- region, and bovine growth hormone poly(A) signal (BGH).
4042 DAVIS ET AL. J. VIROL.

WN virus antigen in the medium was concentrated by precipitation with 10%

PEG-8000. The precipitant was resuspended in TNE buffer (50 mM Tris, 100
mM NaCl, 10 mM EDTA [pH 7.5]), clarified by centrifugation, and stored at 4°C.
Alternatively, the precipitant was resuspended in lyophilization buffer (0.1 M
Trizma and 0.4% bovine serum albumin in borate-saline buffer [pH 9.0]), lyoph-
ilized, and stored at 4°C. Lyophilized preparations were used as the antigen for
the evaluation in MAC- and indirect IgG ELISAs.
WN virus antigen concentrated by PEG precipitation and resuspended in TNE
buffer was extracted with 7.0% ethanol to remove residual PEG (2). Ethanol-
extracted antigens and gradient-purified WN virions were analyzed on a Nu-
PAGE 4 to 12% gradient bis-Tris gel in an Excel Plus electrophoresis apparatus
(Invitrogen Corp., Carlsbad, Calif.) and monitored by electroblotting onto ni-
trocellulose membranes using an Excel Plus blot unit (Invitrogen Corp.). WN
virus-specific protein was detected by Western blot using WN virus-specific
mouse HIAF and MAb 4G2, and NMS was used as a negative serum control (8).
MAC- and indirect IgG ELISAs. The qualities of lyophilized WN virus NRA
produced by the pCBWN-transformed COS-1 cells described in the previous
section were evaluated by ELISAs. One vial of lyophilized NRA, representing

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antigen harvested from 40 ml of tissue culture fluid, was reconstituted in 1.0 ml
FIG. 3. Comparison of Western blot reactivity between NRA pro-
of distilled water and compared with the reconstituted WN virus-infected suck-
duced by pCBWN-transformed COS-1 cells (A) and gradient-purified
ling mouse brain (SMB) antigen provided as lyophilized ␤-propiolactone-inac-
WN virion proteins (V). WN virus-specific mouse HIAF, flavivirus
tivated sucrose-acetone extracts in our facility (9). Coded human specimens were
E-specific cross-reactive MAb 4G2, and eastern equine encephalitis
tested concurrently with antigens in the same test at the developmental stage.
virus monoclonal antibody (EEE MAb) were used at a 1:200 dilution
The MAC- and IgG ELISA protocols employed were identical to the published
in the assay. Sizes are shown in kilodaltons.
methods (18, 24). Human serum specimens were obtained from the serum bank
in our facility, which consists of specimens sent to the division for WN virus
confirmation testing during the 1999 outbreak. In these tests, screening MAC- N. Kumar, M. Godsey, D. Baker, D. Hettler, and C. J. Mitchell, submitted for
and IgG ELISAs were performed on a 1:400 specimen dilution. Specimens publication). Horses were each challenged by the bite of 14 or 15 Aedes albop-
yielding a positive/negative (P/N) optical density (OD) ratio of between 2 and 3 ictus mosquitoes that had been infected by NY99-6425 or BC787 virus 12 days
are considered suspected positives. Suspect serum specimens were tested further prior to horse challenge. Mosquitoes were allowed to feed on horses for 10 min.
in two other tests, ELISA end-point titration and plaque reduction neutralization Horses were examined for signs of disease twice daily. Body temperature was
test (PRNT), for confirmation. Specimens registering P/N ratios of ⱖ3.0 are recorded, and serum specimens were collected twice daily from days 0 (day of
considered positive. infection) to 10 and then once daily through day 14. Pulse and respiration were
Animal vaccination and protection studies. Groups of 10 3-week-old female recorded daily after challenge. The collected serum samples were tested by
ICR outbred mice were used in the study. Mice were injected i.m. with a single plaque titration for detection of viremia and by MAC- or IgG ELISA and PRNT
dose of pCBWN or green flurorescent protein (GFP)-expressing plasmid for antibody response. Vaccinated horses were euthanized by pentobarbital over-
(pEGFP) DNA (Clonetech, San Francisco, Calif.). The plasmid DNA was pu- dose at 14 days after virus challenge and necropsied for gross pathological and
rified from Escherichia coli XL-1Blue cells with EndoFree Plasmid Giga kits histopathological examination, and their carcasses were incinerated within the
(Qiagen) and resuspended in PBS (pH 7.5) at 1.0 ␮g/␮l. Mice that received 100 containment facility.
␮g of pEGFP were used as unvaccinated controls. Mice were injected with the Serological tests. Pre- and postvaccination as well as postchallenge serum
pCBWN plasmid at a dose of 100, 10, 1.0, or 0.1 ␮g in a volume of 100 ␮l. Groups specimens were tested for antibody-binding ability to purified WN virion or
that received 10, 1.0, or 0.1 ␮g of pCBWN were vaccinated by the electrotransfer- recombinant antigen by ELISA, for neutralizing (Nt) antibody by PRNT, and for
mediated in vivo gene delivery protocol using the EMC-830 square-wave elec- antibodies that recognize purified WN virus proteins by Western blotting (8, 27).
troporator (Genetronics Inc., San Diego, Calif.). The electrotransfer protocol PRNT was performed with Vero cells, as previously described (8), using NY99-
was based on the method reported by Mir et al. (25). Immediately following 6480 virus. Endpoints were determined at a 90% plaque-reduction level.
DNA injection, transcutaneous electric pulses were applied by two stainless steel
plate electrodes, placed 4.5 to 5.5 mm apart, at each side of the leg. Electrical
contact with the leg skin was ensured by completely wetting the leg with PBS. RESULTS
Two sets of four pulses of 40 V/mm 25 ms in duration with a 200-ms interval
between pulses were applied. The polarity of the electrode was reversed between Plasmid construct and transient expression of WN virus
the sets of pulses to enhance electrotransfer efficiency. antigen. Expression of the prM and E genes of WN virus was
Mice were bled every 3 weeks following injection, and the WN virus-specific assayed by transfection of plasmid pCBWN into COS-1 cells.
antibody response was evaluated by Ag-capture ELISA and PRNT. Challenge
This plasmid was constructed by inserting the WN virus cDNA
experiments were performed by two methods. Half of the mouse groups were
challenged intraperitoneally (i.p.) at 6 weeks postvaccination with 1,000 50% that encoded the sequence between the prM and E genes into
lethal doses (LD50) (1,025 PFU/100 ␮l) of NY99-6480 virus. The LD50 was the pCBJESS vector to obtain pCBWN (Fig. 2). WN virus-
determined previously by i.p. inoculation of 10-week-old adult ICR mice (data specific protein was detected by IFA on transiently trans-
not shown). The remaining mice were each exposed to the bites of three Culex formed COS-1 cells (data not shown). These cells also secreted
tritaeniorhynchus mosquitoes that had been infected with NY99-6480 virus 7 days
prior to the challenge experiment. Mosquitoes were allowed to feed on mice until
E, prM, and M proteins into the culture medium, that were
they were fully engorged. Mice were observed twice daily for 3 weeks after detected by WN virus HIAF or flavivirus E protein-reactive
challenge. MAb 4G2 in a Western blot analysis. All three proteins had a
Mixed-bred mares and geldings of various ages used in this study were shown similar reactivity and were identical in size to the gradient-
to be WN virus and SLE virus antibody negative by ELISA and PRNT. Four
purified virion E, prM, and M proteins (Fig. 3).
horses were injected i.m. with a single dose (1,000 ␮g/1,000 ␮l in PBS [pH 7.5])
of pCBWN plasmid. Serum specimens were collected every other day for 38 days NRA as an antigen for diagnostic ELISA. An Ag-capture
prior to virus challenge, and the WN virus-specific antibody response was eval- ELISA employing flavivirus group-reactive anti-E MAb 4G2
uated by MAC- or IgG ELISA and PRNT. and 6B6C-1 was used to detect NRA secreted into the culture
Two days prior to virus challenge, 12 horses (4 vaccinated and 8 control) were fluid of pCBWN-transformed COS-1 cells. The antigen could
relocated into a biosafety level 3 containment building at Colorado State Uni-
versity. The eight unvaccinated control horses were the subset of a study that was
be detected in the medium 1 day following transformation, and
designed to investigate WN virus-induced pathogenesis in horses and the poten- the maximum ELISA titer (1:32 to 1:64) in the culture fluid
tial of horses to serve as amplifying hosts (M. L. Bunning, R. A. Bowen, B. Davis, without further concentration was observed between days 2
VOL. 75, 2001 WEST NILE VIRUS DNA VACCINE 4043

TABLE 1. IgM and IgG reactivity to NRA and SMB antigen in a panel of coded human serum specimensa
ELISA titer

Specimen no. IgM IgG Nt antibody

(day postonset) SMB SMB titer
NRA NRA
NY99 virus Eg-101 virus SLE virus NY99 virus Eg-101 virus SLE virus

Positive control 4.19 4.46 5.16 4.54 5.00 4.82 ND

1 (20) 7.42 7.15 6.59 3.74 7.22 6.30 5.12 7.93 1:1,280
2 (14) 4.26 3.12 4.34 1.14 1.14 1.31 1.40 1.8 ND
3 (16) 6.00 5.39 6.18 2.08 1.61 1.85 1.95 2.53 ⬍1:10
4 (30) 3.04 2.23 2.47 1.21 8.48 7.83 6.34 ND 1:160
5 (68) 5.01 5.85 6.28 1.44 1.10 1.14 1.19 1.02 ND
6 (?) 7.30 4.11 3.51 1.11 8.57 8.84 7.76 11.54 1:320
7 (43) 1.90 1.76 2.69 0.79 7.82 7.66 6.40 3.61 ND
8 (20) 1.12 0.98 0.95 0.91 1.53 1.64 1.70 ND ND
9 (3) 1.53 1.37 2.17 1.13 6.88 7.12 6.07 ND 1:5120

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10 (3) 6.43 4.69 6.04 1.87 3.94 4.23 4.38 2.31 ⬍1:10
11 (7) 6.71 4.59 5.78 1.86 3.74 4.30 3.48 2.74 1:20
12 (10) 1.14 1.06 1.17 0.96 6.30 6.59 5.11 7.28 ND
13 (?) 1.13 0.90 1.13 1.33 1.20 1.38 1.41 ND ND
14 (6) 7.43 6.64 8.03 1.71 7.90 7.05 5.44 4.69 1:80
15 (3) 7.44 6.98 8.32 2.89 2.96 3.04 2.43 2.44 1:160
16 (13) 2.88 2.30 4.17 1.01 1.07 1.37 1.47 1.80 ⬍1:10
17 (4) 1.20 0.85 0.90 1.07 0.94 1.34 1.34 1.49 ND
18 (8) 1.09 0.97 1.01 ND 0.67 0.77 0.75 ND ND
19 (73) 1.04 0.96 0.95 ND 0.71 0.80 0.79 ND ND
20 (8) 1.10 1.14 1.01 ND 0.72 0.82 0.82 ND ND
21 (0) 1.06 0.92 0.88 0.91 0.79 0.88 1.11 0.90 ND
Total no. positive 11 11 13 10 10 10
a
SLE virus MAC and IgG ELISAs were conducted as a separate test. Nt antibody titer was assayed in a 90% plaque reduction neutralization test against WN virus.
ND, not done.

and 4. NRA was concentrated by PEG precipitation, resus- negative specimens was positive by SLE virus MAC-ELISA.
pended in lyophilization buffer, and lyophilized for preserva- This result provided further evidence that anti-WN virus IgM
tion. For diagnostic test development, one vial of lyophilized did not cross-react significantly with other flavivirus antigens
NRA was reconstituted with 1.0 ml of distilled water and ti- (39) and was specific to diagnose acute WN virus infection
trated in the MAC- or indirect IgG ELISA using WN virus- regardless of the antigen (NRA or SMB) used in the test. All
positive and -negative reference human sera (18, 24). Dilutions of the specimens were also tested concurrently by indirect IgG
to 1:320 and 1:160 of the NRA were found to be the optimal ELISA, and 10 of 21 specimens were positive with all three
concentrations for use in MAC- and IgG ELISA, respectively. antigens.
These dilutions resulted in a P/N OD450 ratio of 4.19 and 4.54 The two discrepant serum specimens (7 and 9), both from
for MAC and IgG tests, respectively (Table 1). The WN virus the same patient, collected on day ⫺3 and day ⫺43 after onset
SMB antigens produced by the NY99-6480 and Eg101 strains of disease, respectively, were IgM negative with NRA and
were used at a 1:320 and 1:640 dilution for MAC-ELISA and NY99-6480 SMB antigen and suspect for IgM positive to Eg-
a 1:120 and 1:320 dilution for IgG ELISA. The negative con- 101 SMB antigen in the screening test (Table 1). To investigate
trol antigens, PEG precipitates of the culture medium of nor- these two discordant specimens further, six sequentially col-
mal COS-1 cells and normal SMB antigen, were used at the lected specimens from this patient were retested by end-point
same dilutions as the respective NRA and SMB antigens. Hu- MAC- and IgG ELISAs. A greater than 32-fold serial increase
man serum specimens, diluted to 1:400, were tested concur- in the MAC-ELISA titer between days 3 and 15 could be
rently in triplicate with virus-specific and negative control an- demonstrated with all antigens (Table 2). Cerebrospinal fluid
tigens. For the positive test result to be valid, the OD450 for the collected on day 9 after onset of disease also confirmed that
test serum reacted with viral antigen (P) had to be at least this patient indeed was recently infected by WN virus. The
twofold greater than the corresponding OD value of the same cerebrospinal fluid had an IgM P/N reading of 13.71 and 2.04
serum reacted with negative control antigen (N). against Eg-101 and SLE virus SMB antigens, respectively (data
The reactivities of NRA and NY99-0648, Eg101, and SLE not shown). All other specimens had an end-point IgM titer of
virus SMBs were compared by MAC- and IgG ELISAs using 1:1,600 or less with all three antigens when using the absolute
21 coded human serum specimens (Table 1). Of the 21 spec- P/N cutoff of 3.0. Compatible IgG titers were observed with all
imens, 19 had similar results on all three antigens (8 negative three antigens used in the test.
and 11 suspect or positive). Eighteen specimens were also Immunogenicity and protective efficacy of candidate DNA
tested separately using SLE virus SMB antigen. Only 3 of 13 vaccine in ICR mice. ICR mice were immunized by i.m. injec-
Eg-101 SMB-positive specimens were suspect or positive in the tion of pCBWN or pEGPF. The mice were bled 3 and 6 weeks
SLE virus MAC-ELISA (Table 1). None of the WN antigen- after immunization. Individual sera were tested by IgG ELISA,
4044 DAVIS ET AL. J. VIROL.

TABLE 2. Comparison of ELISA titersa obtained with three

antigens on sequential serum specimens from one patient
IgM titer IgG titer
Day
SMB SMB
postonset NRA NRA
NY99 Eg-101 NY99 Eg-101

3b ⬍1:400 ⬍1:400 ⬍1:400 1:1,600 1:3,200 1:3,200

15 1:6,400 ⬎1:12,800 ⬎1:12,800 1:1,600 1:1,600 1:3,200
30 ⬍1:400 ⬍1:400 1:1,600 1:3,200 1:3,200 1:3,200
43c ⬍1:400 ⬍1:400 ⬍1:400 1:3,200 1:3,200 1:3,200
204 ⬍1:400 ⬍1:400 ⬍1:400 1:800 1:1,600 1:1,600
344 ⬍1:400 ⬍1:400 ⬍1:400 1:800 1:1,600 1:800
a
Titers were calculated using an absolute P/N ratio cutoff of 3.0.
b
Random sample 9 in Table 1.
c
Random sample 7 in Table 1. FIG. 4. WN virus-specific reactivity of pre- and postchallenge se-
rum specimens obtained from mice and horses immunized with WN

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virus DNA vaccine. Pooled serum specimens from the mice and horses
used in the experiments were tested at a 1:25 dilution by Western blot
and pooled sera from 10 mice in each group were assayed by analysis using purified WN virion as the antigen. Western blot results
PRNT. All the mice vaccinated with pCBWN had IgG ELISA obtained with pooled horse sera collected before DNA vaccination
titers ranging from 1:640 to 1:1,280 3 weeks after vaccination (week 0), 5 weeks postvaccination or before virus challenge (week 5),
(data not shown). The pooled sera collected at 3 and 6 weeks and 2 weeks postchallenge (week 7). Positive horse serum (lane ⫹) was
from control horse 16, which had PRNT titer of ⬎1,280 on week 4
in the test I group had an Nt antibody titer of 1:80 (Table 3). after virus challenge. Western blot results obtained with pooled mouse
None of the serum specimens from pEGFP control mice dis- sera collected before DNA vaccination (week 0), 3 and 6 weeks post-
played any ELISA or Nt antibody to WN virus. vaccination or before virus challenge (weeks 3 and 6), and 3 weeks
To determine if the single i.m. vaccination of pCBWN could postchallenge (week 9). Mouse HIAF at a 1:250 dilution was used as
the positive control (lane ⫹). Sizes are shown in kilodaltons.
protect mice from WN virus infection, we challenged mice with
NY99-6480 virus either by i.p. injection or by exposure to the
bite of virus-infected Culex mosquitoes. It was evident that the of 1:320 or 1:640 (Table 3). Pooled vaccinated mouse sera re-
presence of WN virus antibody correlated with protective im- acted only with E protein in the Western blot analysis (Fig. 4).
munity, since all mice immunized with WN virus DNA re- Enhancing immunogenicity of the candidate vaccine by elec-
mained healthy after virus challenge (Table 3). All control trotransfer. Mir et al. reported that the local application of
mice developed symptoms of central nervous system infection electric pulses increases the efficiency and reproducibility of in
4 to 6 days later and died an average of 6.9 and 7.4 days after vivo plasmid transfer to muscle fibers after i.m. injection (25).
i.p. or infected-mosquito challenge, respectively. In the vacci- To determine if this unique electrotransfer protocol increases
nated group, the pooled sera collected 3 weeks after virus the immunogenicity of the candidate vaccine, we immunized
challenge (9 weeks postimmunization) had Nt antibody titers groups of 10 mice by this technique with pCBWN (10.0 to 0.1
␮g per animal). At 6 weeks after immunization, all electro-
transfer-immunized groups had a pooled Nt titer equal to or
greater than 1:40 and were completely protected from virus
TABLE 3. Evaluation of protective immunity conferred by
challenge (test II in Table 3). The group immunized by elec-
DNA vaccine for WN virus in mice
trotransfer with 0.1 ␮g of DNA, the lowest dose tested in this
Titerb on wk p.v.: Avg study, had an Nt titer of 1:40, representing only a fourfold
Plasmid
Challenge % Sur- survival
Test and dosea
method c
vival time difference compared with animals receiving 100 ␮g of pCBWN
(␮g) 3 6 9 (days) by i.m. injection. This result suggested that electrotransfer
I pCBWN
could be an alternative immunization protocol to enhance the
100.0 1:80 1:80 1:640 i.p. 100 immunogenicity and protective efficacy of our DNA vaccine.
100.0 1:80 1:80 1:320 Mosq. 100 Immunogenicity and protective efficacy of the candidate vac-
pEGFP (control)
100.0 ⬍1:10 ⬍1:10 i.p. 0 6.9
cine in horses. Four horses were vaccinated i.m. with a single
100.0 ⬍1:10 ⬍1:10 Mosq. 0 7.4 dose of pCBWN and bled every other day prior to infected-
mosquito challenge on day 39. No systemic or local reaction
II pCBWN was observed in any vaccinated horse. Individual horse sera
100.0 1:160 i.p. 100
10.0, E 1:160 i.p. 100 were tested by PRNT. Vaccinated horses developed Nt anti-
1.0, E 1:80 i.p. 100 body titers of ⱖ1:5 between days 14 and 31 (Table 4). Endpoint
0.1, E 1:40 i.p. 100 titers for vaccinated horses 5, 6, 7, and 8 on day 37 (2 days prior
pEGFP (control)
100.0 ⬍1:10 i.p. 0 7.0 to mosquito challenge) were 1:40, 1:5, 1:20, and 1:20, respec-
a
tively. To determine if the DNA vaccine could protect horses
Groups of 10 ICR mice were immunized via a single i.m. injection or i.m.
followed by electrotransfer (E).
from WN virus infection, we challenged vaccinated and unvac-
b
Serum neutralization titers of postvaccination (p.v.) pooled sera were deter- cinated control horses by allowing each horse to be bitten by
mined as described in Materials and Methods. Week 9 sera were collected from approximately 15 virus-infected mosquitoes. Horses vaccinated
the surviving mice 3 weeks after virus challenge.
c
Mice were challenged by i.p. injection of 1,000 LD50 of virus or by exposure with the pCBWN plasmid remained healthy after virus chal-
to the bite of three virus-infected mosquitoes (Mosq.). lenge. None of them developed detectable viremia or fever
VOL. 75, 2001 WEST NILE VIRUS DNA VACCINE 4045

TABLE 4. Serum Nt antibody titers and protective immunity elicited by a single i.m. injection of WN virus DNA vaccine in horses
Titer

Sample (day) Vaccinated horse no. Unvaccinated control horse no.

5 6 7 8 9 10 14 15

Postvaccination
12 ⬍1:10 ⬍1:10 ⬍1:10 ⬍1:10
14 1:10 ⬍1:10 ⬍1:10 ⬍1:10
16 1:10 ⬍1:10 ⬍1:10 ⬍1:10
18 1:40 ⬍1:10 ⬍1:10 ⬍1:10
20 1:40 ⬍1:10 ⬍1:10 ⬍1:10
22 1:40 ⬍1:10 1:10 ⬍1:10
28 1:40 ⬍1:10 1:20 1:10
31 1:40 1:5 1:20 1:10
37 1:40 1:5 1:20 1:20
Postchallenge

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2 1:40 1:5 1:20 1:20 ⬍1:2 ⬍1:2 ⬍1:2 ⬍1:2
4 1:40 1:10 1:20 1:40 ⬍1:2 ⬍1:2 ⬍1:2 ⬍1:2
6 1:40 1:10 1:40 1:40 ⬍1:2 ⬍1:2 ⬍1:2 ⬍1:2
8 1:80 1:20 1:40 1:40 1:80 ⬍1:10 ⬍1:10 ⬍1:10
10 1:80 1:20 1:40 1:80 ⬎1:320 ⬍1:10 ⬍1:10 1:160
12 1:160 1:40 1:80 1:160 ⬎1:320 1:20 1:20 ⬎1:320
14 ⬎1:320 1:40 1:160 1:160 ⬎1:320 1:160 1:10 1:160

Viremia titera range (day) 1.3–2.4 (4) 1.0–1.6 (6) 1.3–2.2 (2)
a
Viremia titer was expressed as log10 Vero cell PFU/ml of serum.

from days 1 to 14. All unvaccinated control horses became proteins into the culture medium of a stably transformed cell
infected with WN virus after exposure to infected-mosquito line. A single i.m. injection of recombinant plasmid DNA in-
bites. Seven of the eight unvaccinated horses developed vire- duced a long-lasting protective immunity and prevented JE in
mia that appeared during the first 6 days after virus challenge. mice (8). The recombinant antigens, formed as EPs that were
Viremic horses developed Nt antibody between days 7 and 9 produced by this stably transformed cell line, also elicited high
after virus challenge. The only horse from the entire study to anti-JE virus Nt antibody (Hunt and Chang, submitted). Our
display clinical signs of disease was horse 11, which became PEG-concentrated recombinant JE virus antigen has also
febrile and showed neurologic signs beginning 8 days after proven to be an excellent alternative to traditional SMB anti-
infection. This horse progressed to severe clinical disease gen used in the diagnostic MAC- and indirect IgG ELISAs
within 24 h and was euthanized on day 9 (Bunning et al., (Hunt and Chang, submitted).
submitted). Four horses, 9, 10, 14, and 15, presenting viremia The same strategy was applied to construct a recombinant
for 0, 2, 4, or 6 days were selected and used as examples in this WN virus plasmid, pCBWN, in the present study. We theo-
study (Table 4). Virus titers ranged from 101.0 PFU/ml of rized that an effective JE virus transmembrane signal peptide
serum in horse 10, the lowest level detectable in our assay, to (TSP) is one of the most important factors that influence
102.4 PFU/ml in horse 9. Horse 14 did not develop detectable downstream protein translocation and topology, thus dictating
viremia during the test period. However, this horse was in-
correct processing of JE virus prM and E proteins by the host-
fected by the virus, as evidenced by Nt antibody detected after
encoded signalase and endopeptidase (8). In the present study,
day 12.
the WN virus-encoded TSP was replaced by JE virus TSP (Fig.
Anamnestic Nt antibody response was not observed in vac-
1). As expected, transiently transformed COS-1 cells expressed
cinated horses, as evidenced by the gradual increase in Nt titer
and secreted prM/M and E proteins into the culture medium,
during the experiment. Existing Nt antibody in the vaccinated
which could be detected by Ag-capture ELISA. Western blot
horse prior to mosquito challenge could suppress initial virus
infection and replication. Without virus replication, the chal- analysis confirmed the presence of three major proteins iden-
lenge virus antigen provided by infected mosquitoes may not tical in size to the E, prM, and M of gradient-purified WN viri-
contain sufficient antigen mass to stimulate an anamnestic im- ons (Fig. 3). More importantly, prM-to-M processing was very
mune response in the vaccinated horse. All vaccinated horses similar between the recombinant antigen and virion protein.
were euthanized 14 days after virus challenge. Gross patholog- The SignalP computer program was used to calculate the
ical and histopathological lesions indicative of WN virus infec- cleavage site (C), signal peptide (S), and combined cleavage
tion were not observed (data not shown). site (Y) scores of JE virus and WN virus TSP by the method of
Nielsen et al. (29). As predicted, SignalP indicated that re-
placement of WN virus TSP for JE virus TSP did not adversely
DISCUSSION
effect the proper signalase cleavage site. Replacement for JE
We previously designed a recombinant eukaryotic expres- virus TSP did increase raw cleavage site (C), signal peptide (S),
sion plasmid that contained an optimal genetic constellation to and combined cleavage site (Y) scores significantly (data not
enhance the expression and secretion of JE virus prM and E shown). More importantly, the most significant predictor of the
4046 DAVIS ET AL. J. VIROL.

TSP (Y) was increased from 0.375 to 0.617. The physical prop- and derived a stably transformed COS-1 cell line, C2, that
erties of the recombinant antigen expressed by plasmid constitutively expresses and secretes recombinant WN virus
pCBWN were not characterized in this study. Physical struc- antigen into the culture medium (G. J. Chang and D. Holmes,
ture and secretion of the antigen expressed by a recombinant unpublished data). The antigen produced by C2 cells has been
plasmid can influence the vaccine potential of flavivirus DNA used to replace the transiently expressed recombinant antigen.
vaccines. The recombinant antigen expressed by plasmid Thus far, 250 vials of recombinant WN virus antigen have been
pCBWN could assemble and form a secreted EP that is very produced and more than 100 vials have been shipped to vari-
similar to the well-characterized JE and tick-borne encephalitis ous states and local health departments for their surveillance
virus antigens (3, 21; Hunt and Chang, submitted). It has been activities. Each vial contains a sufficient amount of antigen to
demonstrated that the plasmid construct encoding an EP of test at least 250 specimens concurrently and in triplicate by
tick-borne encephalitis virus prM and E protein is the most MAC- and IgG ELISAs. Based on our estimation, each vial of
potent vaccine construct of a series of plasmids expressing NRA contains an equivalent amount of antigen produced by
different forms of the E protein (1). This observation shows four-fifths of a suckling mouse brain. Thus, it has great poten-
that our WN virus plasmid has excellent vaccine potential in tial to become the antigen of choice, with a major impact on
mice and horses and supports the notion that a portion of WN the standardization of flavivirus reagents and implementation

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virus prM and E proteins is likely expressed and secreted as of WN virus surveillance in the United States and elsewhere.
EPs. We believe that NRA prepared by our technique could also
The use of the prM-E gene cassette as the flavivirus DNA be used as a biosynthetic subunit vaccine, since feasibility has
vaccine has been reported for different strains of JE virus (8, been demonstrated previously for the antigenically related JE
19, 23) and other flaviviruses, such as SLE virus (31), dengue virus (21; Hunt and Chang, submitted). Currently, we have
serotype 1 and 2 viruses (20, 32, 33), Murray Valley encepha- directed our efforts to using the pCBWN plasmid as a candi-
litis virus (11), and Russian spring-summer encephalitis and date DNA vaccine to prevent WN virus encephalitis in horses.
tick-borne Central European encephalitis viruses (36). In gen- Studies to define an optimal vaccine regimen, long-term im-
eral, vaccine potential, measured by induction of Nt antibody munogenicity and efficacy, and field safety are in progress. As
and protective efficacy by virus challenge, can be improved by an alternative approach, vaccination could be achieved by
multiple i.m., intradermal, or gene gun-mediated intradermal priming the host with DNA, followed by a booster injection
deliveries of DNA vaccine. The gene gun immunization en- with NRA.
hances the uptake of DNA by the professional antigen-pre-
senting cells in the dermis and the opportunity for intracellular ACKNOWLEDGMENTS
processing of in vivo-synthesized antigen directly in these cells We thank Genetronics Inc., San Diego, Calif., for use of the EMC-
(10). However, gene gun immunization requires that plasmid 830 square-wave electroporator and G. Kuno and B. Miller for useful
DNA be applied to the surface of gold beads (12), a step that discussion and advice. We are grateful to D. Hettler and D. Holmes for
could become expensive if multiple vaccine administrations are superb technical assistance.
needed. In addition, the amount of DNA on gold particles that
can be administered in a single application is usually limited to ADDENDUM IN PROOF
2.5 ␮g per mg of gold beads. Studies conducted in monkeys Recently, Konishi et al. (E. Konishi, A. Fujii, and P. W.
with Russian spring-summer and tick-borne Central European Mason, J. Virol. 75:2204–2212, 2001) reported the isolation of
encephalitis virus DNA vaccines have indicated that between 3 a CHO cell line constitutively producing subviral extracellular
and 12 applications per monkey may be require to achieve the particles of JEV only after elimination of the prM processing
effective vaccination dosage (35). site. In the present study, a line of COS cells producing WNV
Electroporation is commonly used to introduce foreign subviral particles with an intact processing site was obtained.
DNA into prokaryotic and eukaryotic cells ex vivo. Recently,
Mir et al. described a similar procedure that uses electric REFERENCES
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