DTIC ADA279844: Dengue Type-2 Virus Envelope Protein Made Using Recombinant Baculovirus Protects Mice Against Virus Challenge

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44
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SAGEN. 73 . REPORT TYPE ..AND DATES COVERED
4. TITLE AND SUBTITLE 5. FUNDING NUMBERS
DENGJE TYPE-2 NTIRUS ENVELTOPE PROTEIN MADE USING RECOMBINANT
BACULOVIRUS PROTECTS. MICE AGAINST VIRUS CHALLENGE
6. AUTHOR(S)
FEIGIINY, ROBERT And BURROUS, JEANNE AND PUTIAK, ROBERT
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PREERPOFORRTM NINUGM BOERRGANIZATION
WALTER REED ARV INSTITUTE OF RESEARCHI
WASHINGION, DC 20307-5100
9. SPONSORING iMONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSORINGMONITORING
AGENCY REPORT NUMBER
U.S. ARMY MEDICAL RESEARCH & DEVEIMOPM(cid:127)MNT CORMAND
FT DETRICK, FREDERICK, MD 21702-5012
11. SUPPLEMENTARY NOTES JljI(cid:127)I(cid:127)LF- Am"I _CTE T . ..
JUN
02
1994
12a. DISTRIBUTION/AVAILABILUTY STATEMENT N- 12b. DISTRIBUTION CODE
NPPROVED FOR PUBLIC RELEASE
DISTRIBUTcON UN IMITED
13. ABSTRACT (Maximum 200 words)
The gene coding for the envelope (E) glycoprotein of dengue 2
virus was cloned into baculovirus (IAutographa californical
nuclear polyhedrosis virus, AcNPV). The recombinant virus
contained the entire E protein gene, preceded by 38 nucleotides
from the end of the prM gene and followed by the first 83
n(Sufc9le) otcideel~lss , ofa bNoSu1t. 1 Wmgh eon f exrepcroemssbeidn anint EI spaondtiogpetne raw afsr umgaidpee rpdearl
lo\9/ cells. This antigen reacted with polyclonal, anti-dengue
type 2 antibody and a dengue type 2-specific, neutralizing
monoclonal antibody. Balb/c mice immunized with the recombinant
antigen produced only non-neutralizing antibody against dengue 2
virus but were partially protected against morbidity and
mortality after intracranial challenge with virulent dengue 2
virus.
14. SUBJECT TERMS 15. NUMBER OF PAGES
DENaJE-2 VIRUS, E PROTEIN, BACULOVIRUS 16. PRICE CODE
17. SECURITY CLASSIFICATION 18. SECURITY CLASSIFICATION 19. SECURITY CLASSIFICATION 20.. LIMITATION OF ABSTI
"OF REPORT OF THIS PAGE OF ABSTRACT
4m. J. rop.M ed. Hvg.. 50(3). 1994. pp. 322-328
Copyright 0 1994 by The Arneican Society of Tropical Medicine and Hygiene
DENGUE TYPE-2 VIRUS ENVELOPE PROTEIN MADE USING
RECOMBINANT BACULOVIRUS PROTECTS MICE
AGAINST VIRUS CHALLENGE
ROBERT FEIGHNY', JEANNE BURROUS. AND ROBERT PUTNAK
Department of Virus Diseases. Walter Reed Army Institute
of Research, Washington, District of Columbia
Abstract. The gene coding for the envelope (E) glycoprotein of dengue-2 virus was
cloned into baculovirus (Autographa californica nuclear polyhedrosis virus). The recom-
binant virus contained the entire E protein gene, preceded by 38 nucleotides from the end
of the prematrix glycoprotein gene and followed by the first 83 nucleotides of nonstructural
protein 1. When expressed in Spodoptera frugiperda (Sf9) cells, approximately I mg of
recombinant E antigen was made per 10' cells. This antigen reacted with polyclonal, anti-
dengue type-2 antibody and a dengue type-2-specific, neutralizing monoclonal antibody.
BALBIc mice immunized with the recombinant antigen produced only non-neutralizing
antibody against dengue-2 virus, but were partially protected against morbidity and mor-
tality after intracranial challenge with virulent dengue-2 virus.
Dengue is an acute disease of viral etiology Positive results have been obtained in the
characterized by fever, headache, body ache, mouse protection model with several recombi-
and rash. Arguably, the most medically impor- nant flavivirus antigens, especially those made
tant arthropod-borne flavivirus, millions of den- in vaccinia and baculovirus." The baculov:"us
Sgue virus infections occur each year in tropical expression system appears especially promaising
4p and subtropical regions of the world, where four for producing subunit protein vaccines because
serotypes are circulating.' 2 this insect virus is not pathogenic for humans
Like other flaviviruses, dengue virus is an en- and has the capability to correctly process most
veloped, positive-strand RNA virus approxi- eukaryotic proteins. We have previously dem-
W
mately 50 nm in diameter. A single open-read- onstrated that mice are protected against Japa-
_
ing frame of approximately 10.5 kilobases (kb) nese encephalitis virus (JEV) and against dengue
__
encodes all viral proteins, which are derived via I virus with recombinant E antigens made in
cotranslational and post-translational proteolytic baculovirus expression systems.? "
1*4
Q6 'processing of a precursor polyprotein. The struc- The aim of the present study was to evaluate
tural capsid (C) protein, the prematrix (prM) in mice a baculovirus-recombinant dengue-2 E
glycoprotein, the matrix (M) protein, and the en- antigen. We found that the recombinant immu-
velope (E) glycoprotein are encoded at the 5' nogen conferred partial protection to mice
end; these are followed by the nonstructural against morbidity and mortality from virulent
(NS) proteins, NSI through NS5.- dengue-2 virus.
There are, at present, no safe and effective
vaccines against dengue. Efforts to develop sub- MATERIALS AND METHODS
unit vaccines have focused mainly on the E gly-
coprotein, the major surface antigen of the viri- Construction of a baculovirus-recombinant
on. It binds to cellular receptors and effects virus dengue 2 E glycoprotein gene and expression
entry by fusion at acidic pH with host cell mem- in Spodoptera frugiperda (Sf9) cells
brane."4 The E antigen contains both T and B
cell epitopes that appear to play an important The RNA genome of dengue 2 virus (PR159
role in immunity," and the isolated, native pro- strain) was cloned and sequenced prior to this
(cid:127)' tein has been demonstrated to subserve protec- study, and a plasmid containing the E gene was
tion in the mouse model."' Therefore E antigen a gift from Dr. James Strauss (California Insti-
may serve as a subunit vaccine. tute of Technology, Pasadena, CA).4 A DNA
restriction fragment containing the complete E
* Deceased. gene was obtained by cutting the plasmid with
322
94 6 1 061
RECOMBINANT DENGUE-2 VIRUS ENVELOPE PROTEIN 323
restriction endonucleases Pie I (cuts at nucleo- Polyacrylamide gel electrophoresis
tide 899) and Eco RI (cuts at nucleotide 2504).
This restriction fragment was treated with bac- Proteins were solubilized in 66 mM Tris, pH
teriophage T4 DNA polymerase and all four 6.8, 1% sodium dodecyl sulfate (SDS), I% glyc-
deoxynucleoside triphosphates to make blunt erol, and 0.66% bromphenol blue at room tern-
ends and cloned into the unique Sma I site of a perature (not boiled), and electrophoresed on
baculovirus recombination vector, pAcMGS, 15% polyacrylamide gels" modified by the in-
which has been described previously."(cid:127)" Re- clusion of SDS in the running buffer but not in
combinant baculovirus was obtained by cotrans- the gel. Molecular weights were determined by
fecting Sf9 cells with the plasmid and wild-type coelectrophoresis of prestained protein standards
baculovirus DNA and then screening for (Bethesda Research Laboratories, Rockville,
polyhedrin-negative plaques. A stock of the re- MD).
combinant virus was used to infect Sf9 cells in
suspension culture at a multiplicity of infection Western blot assay
(MOI) of 10 plaque-forming units (PFU) per
cell. Infected cells were harvested by centrifu- Proteins were analyzed by a nonisotopic mod-
gation at 1,000 X g for 5 min, lysed by Dounce ification of the Western blot procedure,8 using
homogenization, and membrane fractions en- an enzyme-linked second antibody. Briefly, pro-
riched for E protein were prepared from cell lys- teins were separated on polyacrylamide gels and
ates."3 transferred electrophoretically to nitrocellulose
paper. The paper was blocked with 5% casein
Cell cultures, viruses, and antisera in PBS, cut into 0.5 cm-wide strips, and incu-
bated overnight at room temperature with either
The Sf9 cells were propagated and infected HMAF or sera obtained from recombinant-im-
with baculovirus as previously described." "" Ae- munized mice. After the strip was washed with
des albopictis cells (clone C6/36)'` were propa- PBS, biotin-conjugated, goat anti-mouse serum
gated at 28°C in Eagle's minimum essential me- (Sigma, St. Louis, MO) was added and incubat-
dium containing Earle's salts (EMEM; Gibco, ed for 2 hr. The strips were washed and avidin-
Grand Island, NY) and supplemented with 10% biotin-alkaline phosphatase conjugate (1:250 in
fetal bovine serum (FBS, heat inactivated at 5% casein) was added for 45 min. The conjugate
56°C for 30 min), penicillin (100 units/ml), was removed, and after the strip was washed,
streptomycin (100 pýg/ml), 2 mM glutanmine, and substrate (5-bromo-4-chloro-3-indolyi phosphate
0.1 mM nonessential amino acids. For prepara- with nitroblue tetrazolium in dimethylformamni-
tion of dengue-2 virus, C6/36 cells were grown de; Sigma) was added. The reaction was stopped
to 95% confluence in 150-cm2 tissue culture by washing the blots with water after maximum
flasks. Cells were infected with dengue-2 virus color development had occurred.
at an MOI of 0.1 PFU per cell and maintained
in EMEM with 0.6% bovine plasma albumin re- Radioimmunoassay (RIA)
placing FBS. After six to eight days of incuba-
tion, the media was harvested, clarified by cen- The RIA was performed using antigens con-
trifugation at 16,000 X g for 10 min, and viral centrated from dengue-2 virus-infected or un-
antigen in the supernate was concentrated ap- infected C6/36 cell supernates; these were re-
proximately 100-fold by centrifugation at acted with either dengue-2-specific HMAF
100,000 X g for 3 hr at 4°C. Viral antigen, com- (positive control), ascitic fluid from nonimmu-
posed primarily of virion-associated proteins nized mice (negative control), or sera from mice
and NSI, was resuspended in a small volume of immunized with recombinant protein. Antigens
phosphate-buffered saline, pH 7.4 (PBS) and were spotted on nitrocellulose papers and the pa-
stored at -80°C until use. pers were blocked with 5% nonfat dry milk con-
Polyclonal anti-dengue type-2 hyperimmune taining 0.001% sodium azide for 20 min. Sera
A
mouse ascitic fluid (HMAF), and monoclonal or ascitic fluids, diluted 1:500 in PBS, were add-
antibodies (MAbs) 3H5, 4G2, and 2H3 (E anti- ed to the papers and incubated overnight at room
gen-specific) have been characterized previous- temperature. Bound antibody was detected using
ly.(cid:127)6.16 'I-labeled, goat anti-mouse IgG. After the pa-
324 FEIGHNY AND OTHERS
DEN 2 PR59 TABLE I
Reaction of recombinant and authentic dengue-2 en-
c DNA velope (E) antigens with monoclonal and polyclonal
Pie I Eco RI antibodies
IPrM E NSI Antigens
+38 Cleavage +83 Antibodies- Recombinant Et Authentic Et
4G2 1.3(-) +
283 0.6(-) +
3H5 3.2(+) +
Signal added Integration into Baculovirus HNMMAAFF 30..93 ((+- ) )- +
Monoclonal antibody ascitic fluids 4432. 21-3. and 31-15(E antigen-
specific) were used at a 1:250 dilution. Polyclonal anti-dengue-2 hyper-
immune mouse ascitic fluid (HMAF) and normal mouse ascitic fluid
(NMAF) were diluted 1:500.
Expresion in Spodoptero calls t A radioimmunoassay was performed using antigen prepared from
recombinant baculovirns-infected Sf9 cell lysate or control antigen pre-
pared from wild-type baculovirus-infected Sf9 cell lysate containing an
------- - C H equivalent amount of protein. A test was reported as positive (+) if the
counts per mrin (cpm) obtained with recombinant antigen divided by the
+1200 rc2p8me ao btained with control antigen was greater than 2.0.
t A Western blot was performed as described in the Materials and
I .Theen velope (E) E glycoprotein gene Methods with dengue-2 virus from C6636 cells. A positive reaction in-
Fu 1. T ee edicates specific staining of the E protein band.
of dengue-2 (DEN 2) virus expressed in recombinant
baculovirus. Shown (top to bottom) are the restriction
sites used to generate a cleavage fragment that contains juvant, and im injections were given with an
38 nucleotides from the end of prematrix (prM) gly- equal volume of Freund's complete adjuvant for
coprotein encoding a 12-amino acid (aa) hydrophobic
signal sequence, the entire E protein-coding region, the first inoculation (day 0), and with Freund's
and 83 nucleotides (27 amino acids) from the begin- incomplete adjuvant for two boosters (day 7 and
ning of nonstructural protein I (NSI). This restriction day 14). Sera were obtained one day prior to
fragment was cloned into a plasmid transfer vector dengue virus challenge from blood collected
(pAcMGS) and integrated into baculovirus by homol-
ogous recombination. For expression, Spodopterafru aseptically from the retro-orbital plexus with a
giperda (Sf9) cells were infected with the recombinant sterile glass capillary pipette. Mice were chal-
baculovirus. lenged on day 21 (at six weeks of age) by intra-
cranial (ic) injection with l10 PFU (approxi-
mately 100 50% lethal doses) of live,
pers were washed, they were counted in an LKB mouse-adapted dengue-2 virus (NGC strain).
gamma counter (LKB-Wallac, Wallac, Inc., Tur- Mice were observed for up to 24 days for mor-
ku, Finland). bidity and mortality and categorized as 1)
healthy (no apparent signs of infection), 2) ruf-
Virus plaque reduction neutralization 50% fled (the fur had a ruffled look compared with
(PRNTs.) assay normal mice), or 3) paralyzed (slowed gait, par-
tial or complete inhibition of hind limb mobili-
Mouse sera were assayed for dengue-2 virus ty). Data were tested for statistical significance
neutralizing antibody by PRNT assay with a using the chi-square or Fisher's exact tests, as
50% neutralization endpoint.3 appropriate.
Immunization of mice and protection assay RESULTS
Mice (three-week-old, female, BALB/c) were Analysis of the recombinant dengue-2 E protein
immunized by intramuscular (im) and intraperi-
toneal injection with 0.1 ml of baculovirus-in- For expression of the dengue-2 E gene in Sf9
fected Sf9 cell microsomal membrane protein cells, a recombinant baculovirus was constructed
preparation (100,000 X g/hr lysate pellet) en- as shown in Figure 1. Cell lysates from recom-
riched for recombinant dengue-2 E antigen, or binant baculovirus-infected Sf9 cells and con-
an equivalent amount of membrane protein prep- centrated culture media from dengue-2 virus-in-
aration without E antigen (negative control). In- fected and uninfected C6/36 cells were
traperitoneal injections were given without ad- compared by Western blotting with dengue-2 vi-
RECOMBINANT DENGUE-2 VIRUS ENVELOPE PROTEIN 325
ANTIGEN
DEN 2 VIRUS RECOMBINANT PROTEIN UNINFECTED CELLS
(NSD I
Ea--E._ E 7...,
prM.. . c
RECOMS HMAF RECOMB HMAF RECOMS HMAF
ANTISERA
FIGURE 2. Western blot analysis of the baculovirus-recombinant (RECOMB) dengue-2 (DEN 2) envelope
(E) protein. Authentic dengue-2 viral antigens from C6/36 cells (two left lanes), recombinant dengue-2 E antigen
from baculovirus-infected Sf9 cells (two center lanes), and uninfected C6/36 cells (two right lanes) were sub-
jected to polyacrylamide gel electrophoresis and Western blotted with anti-dengue-2 hyperimmune mouse ascitic
fluid (HMAF) or antisera from mice immunized with the recombinant dengue-2 E antigen. Apparent molecular
weights were determined using prestained protein standards that were electrophoresed on adjacent lanes of the
same gel (not shown). Bands indicated are nonstructural protein I (NS 1). (NS I dimer), authentic E protein (E,).
recomabinant E protein (E), protein), and prematrix (prM) glycoprotein.
ms-specific, HMAF, or sera from mice immu- ificity to authentic E antigen6 were reacted in an
nized with the recombinant lysate (Figure 2). RIA with recombinant E antigen. A positive re-
The HMAF reacted with a single protein in the action was seen with one MAb, 3H5, while no
recombinant baculovirus-infected cell lysate that reaction was seen with two others, 4G2 or 2H3
had approximately the same electrophoretic mo- (Table 1).
bility as authentic E protein. Sera from mice im- The recombinant E antigen made in baculo-
munized with the recombinant lysate also react- virus-infected Sf9 cells was detected by RIA in
ed with the same protein and with additional cell lysates but not in extracellular medium, and.
protein bands presumed to be Sf9 cell antigens. therefore, appeared to be cell-associated. The
The HMAF, but not sera from recombinant bac- yield of recombinant E antigen was estimated to
ulovirus-immunized mice, reacted with authentic be approximately I mg per 10' cells by corn-
dengue-2 virus proteins, NSl dimer (-70 kD), paring the intensity of bands on a Western blot
E (-60 kD), and prM (-20 kD). assay of recombinant-infected cell lysate and
To verify the antigenic identity of the recom- highly-purified E antigen of known concentra-
binant E protein, Mabs with demonstrated spec- tion.
326 FEIGHNY AND OTHERS
TABLE 2 recombinant protein administered in three equal-
Antibody response of mice immunized with baculovi- volume doses. After immunization, the mice
rus-recombinantd engue-2 envelope (E) antigen* were bled and sera were tested for virus-binding
Total antigen and neutralizing antibody against dengue-2 vi-
dose (ILg) RIA (P/N)It PRNTJ rus. Sera from recombinant-immunized mice
1.5 7.4 <10 showed antibody that bound to dengue virus an-
9.0 13.5 <10 tigen, with the highest level seen in mice im-
Rato by4.r0d a amunized with the highest dose of recombinant
duc•tiRoena cntieount rabliyz artaiodnio i(mPRmNunTo) aassssaayy (oRfI Ase)r a afnrdo md emngiucee ivmirmusu npilzaeqdu ew-rieth- antigen. However, none of the sera had measur-
different doses of recombinant E antigen.
t Positivelnegative (P/N) = counts per min (cpm) ohained with anti- able titers of anti-dengue-2 virus-neutralizing
gen prepared from dengue-2 virus-infected C6/36 cell supemates, divided antibody (Table 2).
by cpm obtained with uninfected C6/36 cell supemates used at an ap-
proximately equal total protein concentration. Mouse sera were used at Immunized mice were then challenged with
a 1:500 dilution. P/N values >2.0 were considered positive.
t Virus PRNT assay with a 50% endpoint (PRNT.). Values of tO or live dengue-2 virus and observed for morbidity
greater were considered positive. <10 negative. and mortality (Table 3 and Figure 3). The num-
ber of mice that survived challenge was one of
trecombinant 10 in the control group, five of 10 in the group
Immunization of BALB/c mice with romia tha eevd15~~ frcmiatatgn(
dengue 2 E antigen that received 1.5 pg of recombinant antigen (P
= 0.05, by Fisher's exact test), nine of 10 in the
To test the recombinant E protein as a protec- group which received 9 P±g (P = 0.0005), and
tive immunogen, groups of containing 10 mice six of 10 in the group that received 19.5 p±g (P
each were immunized with 1.5, 9, or 19.5 ;.g of 0.03). Although the protection rate decreased
IO0
so-
70
1 6
I:I
30
20-
10-
I0-
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
DYS OF O 5110MlN
FIGURE 3. Survival of mice immunized with recombinant dengue-2 envelope (E) antigen following challenge
with live dengue-2 virus. Groups of 10 mice each were immunized with 1.5 g±g (0), 9 pig (+), and 19.5 ltg (0)
of baculovirus-recombinant dengue-2 E antigen or with an equivalent amount of protein from baculovirus-
infected Sf9 cells that did not contain E antigen (A). Following immunization, mice were challenged by intra-
cranial injection with approximately 100 50% lethal doses of dengue-2 virus (NGC strain). The percentage of
mice surviving at each day postchallenge is shown.
327
RECOMBINANT DENGUE-2 VIRUS ENVELOPE PROTEIN
somewhat in the latter group, this was not sta- TABLE 3
tistically significant (P = 0.15). Effect of vaccination with recombinant envelope anti-
Immunized mice also showed some protection gen on dengue morbidity in mice
against morbidity that was significant, however, Length
this was observed only in the group that received Wodfa ytsim)f reom
9 jig of recombinant E antigen (P = 0.03, by o, Day of onset of th of o,
Doeo 'tof illtness Lengthof Dyf
chi-square test). In this group, the day of onset ifg) illness. to death illlnesst death
of illness as determined by appearance of ruffled 0 5.5 3.5 14.5 (I) 9.6
fur or slowed movement was delayed (day 9.6 1.5 6.5 3.8 9 (5) 10
versus day 5.5 in controls), the length of illness 9.0 9.6 8 5.4 (9) 20
was decreased in those mice that survived chal- 19.5 7.7 3 9.5 (6) 9.5
lenge (5.4 days versus 14.5 days in controls), groutDp.ay of onset of illness is the mean determined for all mice in each
and the mean time to death was increased in t Length of illness is the mean number of days of illness determined
for those mice that ultimately survived challenge and recovered. The
nonsurvivors (8 days versus 3.5 days in con- number of survivors is shown in parentheses.
trols).
other than antibody play a crucial role in pro-
DISCUSSION tection. One explanation is that T cell epitopes
A 1.6-kb dengue-2 cDNA restriction fragment play a large role. Unlike most B cell epitopes,
encoding a 12-amino acid hydrophobic signalupon secondary struc-
peptide at the end of the prM protein, the entire ture,' most helper and cytotoxic T cell epitopes
E protein, and the first 27 amino acids of NS I appear to be definable by primary structure
protein was cloned and expressed using a re- alone22, and therefore may not be so dependent
combinant baculovirus. The recombinant protein upon antigen conformation. Additional studies
need to be done to test this hypothesis and to
reacted with anti-dengue-2 HMAF and with a elucidate other factors that may subserve protec-
dengue-2-specific, neutralizing MAb (3H5) that tion.
recognizes a linear epitope on E protein.t9 How-
ever, the recombinant protein failed to react with Acknowledgments: We gratefully acknowledge John
two other E-specific MAbs (4G2 and 2H3). Hendricks, Jr., Martin Lackovic, Diane Elgin, and
These MAbs appear to recognize epitopes that Kristina Strupczewski for assistance with animal han-
are more conformation-dependent20 (unpubli- dling and serologic assays.
shed data), suggesting that significant differ- Financial support: This work was supported in part by
ences in folding exist between the recombinant Department of Defense (DOD) grants DAMD17-C-
2237, DAMDI7-C-6156, and DAMDI7-C-6612 to
and the native protein. MicroGeneSys, Inc. (Meriden, CT) for production of
When used to immunize mice, the recombi- the recombinant baculovirus used in this study.
nant E protein stimulated, in a dose-dependent Disclaimer: The views presented are those of the au-
manner, the production of antibody that bound thors and should not be construed as official policy
to authentic dengue-2 virus in an RIA. However, positions of the Department of the Army or the DOD.
this antibody failed to neutralize dengue-2 virus Authors' address: Jeanne Burrous and Robert Putnak.
in vitro. Interestingly, although the recombinant Department of Virus Diseases, Walter Reed Army In-
E antigen failed to stimulate dengue virus-neu- stitute of Research, Building 40, Room 2036, Wash-
tralizing antibody, immunized mice were par- ington, DC 20307-5100.
tially protected (up to 90%) against death from
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