M and E proteins are protected from lethal JEV infection

VIROLOGY

188, 714-720

(19%)

Mice Immunized with a Subviral Particle Containing the Japanese Encephalitis Virus prM/M and E Proteins Are Protected from Lethal JEV Infection EIJI KONISHI,* STEVEN PINCUS,t ENZO PAOLElTl,t ROBERT E. SHOPE,” THOMAS BURRAGE,+ AND PETER W. MASON*+’ *Yale Arbovirus Research Unit, Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, Connecticut 065 10; t Virogenetics Corp., 465 Jordan Road, Rensselaer Technology Park, Troy, New York 12 180; and *Plum Island Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Greenport, New York 11944 Received January 6, 1992; accepted

February 24, 1992

Extracellular subviral particles produced by HeLa cells infected with a recombinant vaccinia virus encoding the prM and E genes of Japanese encephalitis virus (JEV) were purified and characterized. These particles contained the JEV prM/M and E proteins embedded in a lipid bilayer, and RNA was not detected in particles using the polymerase chain reaction and primers recognizing a part of the JEV E gene. The particles were uniformly spherical with a 20-nm diameter and had 5-nm projections on their surface. Mice that received a single inoculation of the purified extracellular particles emulsified with Freund’s complete adjuvant were fully protected against 4.9 X lo5 LD, of JEV. Comparison of the neutralizing and hemagglutination-inhibiting antibody titers and radioimmunoprecipitation data showed that immunization with the particles induced an immune response similar to that following inoculation with the recombinant vaccinia Virus.

0 1992 Academic

Press,

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INTRODUCTION

to M, presumably by a cellular protease located in the secretory pathway, and M appears to be the predominant species present in extracellular virus particles although some uncleaved prM is also present (Chambers et al., 1990). Our evaluations of recombinant vaccinia viruses have supported these proposed cleavages by showing that recombinant viruses encoding only prM and E of JEV induce the synthesis of correctly processed forms of prM, M, and E (Konishi et al., 1991). Several investigators have developed flavivirus vaccine candidates using recombinant vaccinia virus vectors (Zhao et al., 1987; Deubel et al., 1988; Haishi et al., 1989; Bray et al., 1989; Falgout et al., 1990; Hahn et al., 1990; Putnak and Schlesinger, 1990; Yasuda et al., 1990; Men et a/., 1991; Bray and Lai, 1991). Analyses of recombinant vaccinia viruses expressing portions of the ORF of JEV (Mason et a/., 1991; Konishi et al., 1991) and yellow fever virus (Pincus, et al., 1992) have shown that recombinants that induce the synthesis of extracellular particles containing prM/M and E elicited high levels of neutralizing (NEUT) and hemagglutination-inhibiting (HAI) antibodies and protected against lethal flavivirus challenge. Furthermore, recombinant viruses that produce intracellular (but not extracellular) forms of E provided lower levels of antibodies and protection. We concluded from these data that the ability of recombinant vaccinia viruses to produce extracellular particles in vitro was related to induction of protective immunity in inoculated animals.

Members of the family Flaviviridae including Japanese encephalitis virus (JEV) are pathogens of medical and veterinary importance @hope, 1980; Monath, 1986). Although some flavivirus vaccines have been successfully produced using traditional technologies, there is a need for new and improved vaccines, which may be engineered by recombinant technology (Brandt, 1988). The current JEV vaccines for human use are formalin-inactivated virion fractions purified from JEV-infected mouse brain (Takaku er al., 1968). Although these vaccines are substantially free from murine encephalitogenic basic proteins, allergic reactions in humans have been reported (Andersen and Rernne, 1991). Furthermore, the relatively high cost for production from mice and the safety for personnel dealing with large quantities of highly infectious material has raised concern. The flavivirus virion contains an envelope glycoprotein (E), a membrane protein (M), and a capsid protein (C). These three proteins are synthesized in the order C, M, E at the start of a single long open reading frame (ORF) encoded by the flavivirus genome. The M protein is found in infected cells as a glycosylated precursor (prM), and prM and E appear to be released from the nascent polyprotein by cotranslational cleavage by signal peptidase. Late in virion maturation, prM is cleaved ’

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PROTECTION BY A JE SUBVIRAL PARTICLE

In this study, we purified and characterized the extracellular particles produced from HeLa cells infected with a recombinant vaccinia virus, vP829, encoding the JEV prM and E genes. In addition, we evaluated the immunogenicity of these extracellular particles in mice to understand further their role in eliciting protective immunity and to evaluate their usefulness as a flavivirus vaccine candidate.

MATERIALS AND METHODS Cell lines and virus strains HeLa cells (ATCC CCL2) were grown at 37” in Eagle’s minimal essential medium (MEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), nonessential amino acids (NEAA), and antibiotics. VERO (ATCC CCL81), CHO-Kl (ATCC CCLGl), CER (Smith et al., 1977), and SW-1 3 (ATCC CCL1 05) cells were grown under the same conditions except using 5% FBS without NEAA. C6/36 cells (Igarashi, 1978) were grown under the same conditions as HeLa using twice as much NEAA. The JEV viruses, Nakayama and P3, used in this study have been described (Mason, 1989; Mason et al., 1991). The stock of the vaccinia recombinant virus, VP829 (Konishi et al., 1991), was prepared in HeLa cells.

Purification of virions and extracellular particles HeLa cells were infected with VP829 at a m.o.i. of 2 and then grown for 24 hr at 37” in MEM containing 1o/o FBS. The clarified culture fluid was then centrifuged for 15 hr at 25,000 rpm in a Sorvall AH629 rotor at 4” onto a sucrose cushion made with 0.5 ml of 5% sucrose and 0.5 ml of 20% sucrose (w/w, prepared with TN buffer, 10 mn/lTris-HCI, pH 7.5, 100 mn/r NaCI). Afraction from the interface between the 5% and 20% sucrose was further concentrated by ultrafiltration (with either Ultrafree cellulose membranes (Millipore Corp., Bedford, MA) or YM membranes (Amicon, Beverly, MA)). After clarification, the concentrate (2 ml) was applied to a 1O-35% continuous sucrose gradient (w/w, prepared with TN buffer) and centrifuged for 3 hr at 36,000 rpm in a Sorvall TH641 rotor at 4”. Fractions were collected and stored at -70”. HA activity was used to define the purified VP829 extracellular particle fraction. JEV virions were purified in a similar manner from culture fluids harvested 48 hr postinfection from VERO cells infected at a m.o.i. of 5 with the Nakayama strain except that the initial step gradient consisted of 10 and 65% sucrose.

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Sodium dodecyl sulfate-containing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting Samples were mixed with electrophoresis sample buffer containing dithiothreitol at a final concentration of 50 mM and run on SDS-containing polyacrylamide gels as described by Laemmli (1970). E protein concentration was estimated by comparison with bovine serum albumin (BSA) samples in Coomassie brilliant blue-stained gels, and silver staining (Merril et a/., 1981) was performed with the Bio-Rad Silver Stain kit according to the manufacturer’s instructions (Bio-Rad Laboratories, Richmond, CA). Proteins were transferred to a nitrocellulose membrane and immunochemitally detected with monoclonal antibodies (MAbs) against E (J3-11 B9; Mason et a/., 1989) or prM/M (J22Fl; Mason et al., 1987) or polyclonal anti-JEV antibodies obtained in the form of hyperimmune mouse ascitic fluid (HMAF; Brandt eta/., 1967) and stained with [1251]labeled antimouse IgG (Amersham, Arlington Heights, IL) as described by Towbin et al. (1979).

Treatment with Triton X-l 00 (TX1 00) Purified extracellular particles were diluted in TN buffer in the presence or absence of TX1 00 (final co Icentration of 1 mg/ml), and then applied to a 5-20% continuous sucrose gradient (w/w) prepared with or without 1 mg/ml TXlOO. Both gradients were centrifuged for 3 hr at 35,000 rpm in a Sotvall TH641 rotor at 4”, and fractions were examined for JEV prM/M and E antigenic reactivity by direct enzyme-linked immunosorbent assay (ELISA; see below).

Direct ELISA Samples were diluted in 0.1 M sodium carbonate buffer (pH 9.6) and used to coat 96-well microplates (Immulon 4, Dynatech Laboratories, Inc., Chantilly, VA) by overnight incubation at 4”. After blocking with BSA, the solid phase was reacted with anti-M (prM) or E MAbs (described above; 1:500 dilution) and then probed with an alkaline phosphatase-conjugated antimouse IgG (H- and L-chain specific; Bio-Rad Laboratories, Richmond, CA; 1:500 dilution). The enzyme activity on the solid phase was then detected with p-nitrophenyl phosphate at 2 mg/ml, and the reaction was measured at 414 nm.

HA test and plaque assay Hemagglutination (HA) tests were performed by a modification of the method of Clarke and Casals (1958). Virus titers were determined on VERO cell

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KONISHI ET AL

monolayers 1991). Detection

as previously

described

of RNA in extracellular

(Konishi

et al.,

particles

The polymerase chain reaction (PCR) method was used to determine if nucleic acid containing the E coding region of JEV was present in the extracellular particles using JEV RNA extracted from purified virions as a standard. First-strand cDNA synthesis was primed with random hexamer oligonucleotides using Moloney murine leukemia virus reverse transcriptase (BRL Life Technologies, Inc., Gaithersberg, MD) following the manufacturer’s recommendation. PCR was performed by the method of Saiki et al. (1988) with two primers recognizing nucleotides 2221-2244 and nucleotides 2369-2397 of the JEV ORF, using amplitaq polymerase (Perkin-Elmer Cetus, Norwalk, CT) and the following thermal steps: 94” for 1 min, 45” for 1 min, and 72” for 2 min for 30 cycles and a final extension for 6 min at 72”. The PCR reactions were then examined for the presence of the 177-bp product following electrophoresis in 4% NuSieve agarose (FMC BioProducts, Rockland, ME) gels and staining with ethidium bromide. Electron

microscopy

Fractions containing the purified extracellular particles and the binary ethylenimine (BEl)-inactivated JEV virions were processed for negative staining by adsorption to Formvar-coated nickel grids. After adsorption, the grids were either stained directly with 2% phosphotungstic acid (PTA; pH 7.5) or fixed for 10 min with 2% glutaraldehyde and stained with 2% uranyl acetate (UNAc; pH 4.5). Animal protection

experiment

The small amount of infectious vaccinia virus that remained in the purified extracellular particle fraction was inactivated by incubating the fraction at 28” for 6 hr with freshly prepared BEI at a final concentration of 0.06% (Bahnemann, 1990). The BEI was neutralized with sodium thiosulfate and the fraction was used to inoculate mice. Groups of 3-week-old outbred Swiss mice were given a single inoculation of extracellular particles or recombinant vaccinia virus, vP829. The extracellular particle fraction was either emulsified with Freund’s complete adjuvant and administered subcutaneously (SC; 10 pg or 1 pg of E) or by intraperitoneal (ip) injection without adjuvant (1 pg of E). Three weeks later, sera were collected and mice were challenged by ip injection with 4.9 x 1O5 LD,, of the Beijing P3 strain of JEV (Huang, 1982). Following challenge, mice were observed daily for 3 weeks.

FIG. 1. Silver-stained gels and Western blots comparing purified JEV virion (V) and extracellular particle (EP) fractions. 26,000 HA units of each sample were applied per lane to a 15% polyacrylamide gel and following electrophoresis a portion of the gel was fixed and silver stained (protein), and the remainder was transferred to a nitrocellulose membrane (see Methods). Following transfer, portions of the blot were reacted with HMAF, MAb to E, or MAb to prM/M followed by decoration with [‘251]-anti-mouse IgG (see Methods).

RESULTS Purification

of extracellular

particles

The yield of extracellular particles was compared among CER, CHO-Kl, HeLa, VERO, and SW-l 3 cell lines by monitoring HA activity 24 hr following infection with VP829 at a m.o.i. of 2. HeLa and SW-13 showed the highest HA titer in clarified culture fluids, and HeLa were chosen for all future analyses since less cytopathic effect was caused by the recombinant virus, thus limiting potential release of cellular components into the HA-containing culture fluid. The recovery of HA activity in the purified fraction was 70-80%, and more than 99% of the infectious vaccinia virions released from cells were eliminated during the purification process. Based on estimates from Coomassie blue staining (not shown), extracellular particles containing approximately 10 pg of E (1 014 molecules, 2 X lo5 HA units) were released from lo7 vP829-infected HeLa cells. Biochemical particles

characterization

of extracellular

The purified extracellular particle fraction was compared with JEVvirion preparations by silver staining and Western blotting (Fig. 1). The Western blots revealed that the virion fraction and the extracellular particle fraction both contained prM, M, and E, whereas only

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PARTICLE

and stained with UNAc (Fig. 3) but PTA-stained mate-

1.0

rial showed similar topography and size distribution. The extracellular particles ranged in diameter from 15 to 25 nm with an average of 20 nm. Short (5 nm) projections radiated uniformly from the surface of the partcle, similar to those observed in JEV virions (Kitano et a/., 1974). lmmunoelectron microscopy showed that the extracellular particles that were reacted with a MAb to E but not with a MAb to NSl could be decorated with colloidal gold-conjugated anti-mouse lg or Protein G (data not shown).

no triton

0.6 ??

0.4 i

0.2

lmmunogenicity

‘k&

0.0 m 0.6 1

IO FRACTION NUMBER

20

FIG. 2. Profile of prM/M and E antigenic reactivity in sucrose density gradients prepared from purified extracellular particles with or without TX1 00 treatment.

the virion fraction contained C. The silver-stained gel showed that the E protein was the major protein component of the extracellular particle fraction that also contained clearly visible amounts of M. The extracellular particle fraction also contained a protein that comigrated with BSA (not visible in Fig. 1) and two proteins of approximately 1O-l 1 kDa, which migrated ahead of C (Fig. 1). Purified extracellular particles were analyzed on a 5-200/o sucrose density gradient with or without TX1 00 treatment. The presence of prM/M and E in each fraction was determined by the direct ELISA using MAbs. As shown in Fig. 2, prM/M and E comigrated in each gradient and the peak obtained under the TX1 00-free condition was shifted to the top of the gradient following treatment with TX1 00, indicating that the extracellular particles were membrane vesicles containing prM/M and E. JEV nucleic acid was not detected in the purified extracellular particles corresponding to 1 pg of the E protein under the PCR conditions employed. Judging from the detection limit using intact JEV RNA extracted from purified virion as a standard, the amount of RNA present in the extracellular particles containing 1 pg of E (1013 molecules) was calculated to be less than lo6 molecules. The absence of RNA was consistent with both the mobility of the particles in sucrose density gradients and the absence of JEV genomic length RNA from vP829-infected cells.

Ultrastructural particles

characterization

of extracellular

The morphology of the extracellular particles was best observed in fractions fixed with glutaraldehyde

of extracellular particles

NEUT and HAI antibody titers in sera pooled from all mice in each group prior to challenge showed that inoculation with extracellular particles containing 10 hg of E elicited levels of antibody similar to those elicited by 1O7 PFU of VP829 (Table I). Even mice immunized with one-tenth of this amount of extracellular particles (corresponding to 1 pg of E) without adjuvant showed a detectable NEUT antibody titer. The immunogenicity of the extracellular particle preparation was further supported by the ability of these prechallenge sera to immunoprecipitate radiolabeled JEV E antigen (Fig. 4). The prechallenge sera obtained from animals inoculated with the BEI-treated extracellular particles did not immunoprecipitate any vaccinia virus antigens (data not shown), demonstrating that BEI treatment had completely eliminated residual vaccinia viruses from the extracellular particle fraction. Furthermore, groups of mice injected with 3.3 X 1O* PFU of VP829 did not show any detectable anti-JEV antibodies, confirming that the immune responses detected in the animals inoculated with the extracellular particles were not due to infection by residual recombinant vaccinia virus. All of the mice immunized with extracellular particles containing 10 pg of E were fully protected from the lethal JEV challenge, demonstrating that this dose of inactivated antigen could provide the same level of protection as inoculation with 1O7 PFU of VP829 (Fig. 5). Significant protection was also observed with a single inoculation of extracellular particles containing 1 pg of E emulsified with adjuvant. Mean survival time of mice immunized with the same dose of extracellular particles without adjuvant (6.8 days) was longer than that of control mice injected with adjuvant alone (5.0 days). Although the differences are not statistically significant (P> 0.05) they are consistent with the fact that some NEUT antibodies were obtained in these animals.

DISCUSSION The development of second-generation vaccines for flavivirus diseases has focused on expression of the

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KONISHI ET AL

FIG. 3. Electronmicrograph

showing

negatively

stained purified extracellular

relevant immunogens in vaccinia viruses or baculoviruses. In several cases, investigators have sought to produce extracellular forms of E by engineering carboxy-terminally truncated E proteins (Deubel et a/., 1991; Men et a/., 1991; Putnak et al., 1991). These techniques should enable the production of extracellular forms of E, an important criteria for production of pure immunogens. The recombinant vaccinia viruses that we engineered for producing extracellular partcles (Mason et al., 1991; Konishi et al., 1991) provide another method for producing a subunit vaccine candi-

particles.

Bar indicates

100 nm

date. These particles are likely to present the E protein in a more natural state than the truncated forms of E, since the extracellular E appears identical to E found in the virion and the particles also contain prM/M. The extracellular particles produced by vP829-infected cells are membrane vesicles of 20 nm diameter containing the JEV prM/M and E proteins embedded in a lipid bilayer. The extracellular particles are similar to SHA particles released from JEV-infected cells in behavior on sucrose density gradients (Mason et a/., 1991; Konishi et a/., 1991). In the process of flavivirus

TABLE 1 COMPARISONOF IMMUNE RESPONSESTO VACCINIA RECOMBINANTVP829 AND THE RECOMBINANT-DERIVEDEXTRACELLU~ARPARTICLES Antibody titer lmmunoger? Particles Particles Particles Buffer VP829 VP829

Quantityb

Adjuvant”

1oPg 1 &I 1 &I

+ + + -

1.0x 1O’PFU 3.3 x 10’ PFUg

Injection routed SC SC ip SC ip ip

Number of mice

NEUT”

HAI’

4 5 5 5 5 5

1:80 1:20 1:lO <1:10 1:80
1:40 1:lO
B Groups of 3-week-old male mice were immunized with the purified extracellular particles (Particles), buffer, or a recombinant vaccinia virus (vP829). b Amount of extracellular particles expressed as the quantity of E protein (10 pg of E corresponded to 2 X 1O5 HA units) or infective titers for vP829. ’ Freund’s complete adjuvant. ‘Subcutaneous (SC) or intraperitoneal (ip). e Serum dilution yielding 90% reduction in plaque number. ‘Serum dilution. B The amount of vaccinia virus contained in the putified extracellular particle fraction corresponding to 10 rg of E before inactivation with BEI.

PROTECTION

BY A JE SUBVIRAL

maturation in vertebrate cells, some portions of cellular (endoplasmic reticulum; ER) membranes in which the prM and E proteins are anchored are assembled with the core composed of viral genomic RNA and the C protein and finally are exocytosed as infectious virions (Chambers et al., 1990). It is likely that SHA particles are derived from membranes containing prM and E, which assemble without the core. In vP829-infected cells, prM arid E are properly synthesized and accumulate in the ER in a membrane-bound form identical to that observed in JEV-infected cells (Konishi et al., 1991). Furthermore, these vaccinia recombinant-encoded proteins assemble into a mature SHA-like partcle in the absence of the other components (viral genomic RNA and C) required to produce a flavivirus virion. Our present study demonstrates that the particles alone are immunogenic and protect mice from lethal JEV challenge and support our hypothesis that these particles provide a key ingredient in the protective immune response elicited by inoculation with our recombinant vaccinia viruses (Mason et a/., 1991; Konishi et a/., 1991). The ability to obtain high yields of purified extracellular particles probably reflects their stability. Preliminary stability studies have shown no detectable decrease in HA activity following incubation at 4” for 7 days or 28” for 3 days in PBS containing 0.1 Yo BSA. Their stability may be.important for long-term storage and transport, an important requirement for a flavivirus subunit vaccine. The immunogenicity and stability of these particles raise the prospect that they, alone or in combination with live vectors, could be useful in the development of flavivirus vaccines.

NSl’ E

NSl

FIG. 4. Analysis of the E protein-specific reactivity of prechallenge sera from mice inoculated with the purified extracellular particles or the recombinant vaccinia virus, vP829. Sera collected from all the animals used in the protection experiments were pooled and aliquots were tested for their ability to immunoprecipltate [35S]-labeled E harvested from the culture fluid of JEV-infected cells (Mason era/., 1991). The two lanes on the left side show the positions of the E and NSl proteins precipitated with monoclonal antibodies.

PARTICLE

719

DAYS POST CHALLENGE FIG. 5. Survival data for groups of mice inoculated once with purified extracellular particles containing 10 pg of E with Freund’s complete adjuvant (open circle), particles containing 1 pg of E with adjuvant (closed circle) or without adjuvant (open triangle), adjuvant alone (closed triangle), 10’ PFU of VP829 (open square), or 3.3 X 10’ PFU of VP829 (closed square) and then challenged with 4.9 X lo5 LD,, of JEV.

ACKNOWLEDGMENT We thank Dr. Mary Kay Gentry (WRAIR, Washington, D.C.) for ascitic fluids produced from hybridomas J2-2Fl and J3-1 1 B9. This work was supported by grants from the National Institutes of Health, Al10987-17, the US. Army Medical Research and Development Command, DAMDl7-90-Z-0020. and The National Science Foundation, DMB 8515345. E.K. was supported by the Department of Medical Zoology, Kobe University School of Medicine, Kobe, Japan. We would like to thank Dr. B.A.L. Fonseca (Yale University School of Medicine) for helpful suggestions and contributions to various asoects of this work.

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