Recombinant vaccinia virus producing the prM and E proteins of yellow fever virus protects mice from lethal yellow fever encephalitis

VIROLOGY

187, 290-297

Recombinant

(1992)

Vaccinia Virus Producing the prM and E Proteins of Yellow Protects Mice from Lethal Yellow Fever Encephalitis

Fever Virus

STEVEN PINCUS,* PETER W. MASON,++ EIJI KONISHl,t BENEDITO A. L. FONSECA,t ROBERT E. SHOPE,t CHARLES M. RICE,5 AND ENZO PAOLElTI**’ *Virogenetics Corporation, 465lordan Road, Rensselaer Technology Park, Troy, New York 12180; tYale Arbovirus Research Unit, Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, Connecticut 06510; *Plum Island Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Greenport, New York 11944; and BDepartment of Molecular Microbiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63 11 O- 1093 Received October 9, 199 1; accepted

November

2 1, 199 1

Four recombinant vaccinia viruses were constructed for expression of different portions of the 17D yellow fever virus (YFV-17D) open reading frame. A recombinant, vP869, expressing prM and E induced high titers of neutralizing and hemagglutination inhibiting antibodies in mice and was protective against intracranial challenge with the French neurotropic strain of YFV. Levels of protection were equivalent to those achieved by immunization with the YFV-17D vaccine virus. Recombinant vaccinia viruses expressing E and NSl, C prM, E, NSl, or only NSl failed to protect mice against challenge with YFV despite eliciting antibodies to NSl. The vP869infected HeLa cells produced a particulate extracellular hemagglutinin (HA) similar to that produced by YFV-infected cells, supporting previous studies with Japanese encephalitis virus (Mason eta/., 1991), suggesting that the ability of recombinant vaccinia virus to produce extracellular HA particles is important for effective flavivirus immunity. 0 1992 Academic Press, Inc.

INTRODUCTION

Gould et a/., 1986; Kaufman et al., 1987, 1989; Henchal et al., 1988; Kimura-Kuroda and Yasui, 1988; Mason et a/., 1989). In the case of MAbs to E, passive protection correlated with in vitro neutralizing activity, but weakly neutralizing prM MAbs or nonneutralizing antibodies specific for prM or NSl were also found to afford passive protection. Schlesinger and co-workers provided additional evidence that NSl immunity could protect animals from infection by demonstrating that purified NSl from yellow fever-infected cells (Schlesinger et al., 1985, 1986) or dengue type 2 virus-infected cells (Schlesinger eta/., 1987) could protect animals from infection with the homologous virus. Knowledge of flavivirus genome structure has led to the application of recombinant techniques to the production of new vaccine candidates. These approaches have included purified Escherichia co/i fusion proteins (Cane and Gould, 1988; Mason et al., 1989), crude lysates from moth cells infected with recombinant baculovirus (Zhang et al., 1988; Matsuura et a/., 1989; McCown et al., 1990; Deubel et al., 1991; Shiu et al., 1991; Despres et al., 1991), and live recombinant vaccinia viruses (Deubel et a/., 1988; Zhao et a/., 1987; Haishi et al., 1989; Bray et al., 1989; Falgout et al., 1990; Hahn et a/., 1990; Putnak and Schlesinger, 1990; Yasuda et al., 1990; Mason et a/., 1991; Men et a/., 1991). Several of these, notably the recombinant baculovirus and vaccinia approaches, have yielded promising efficacy data, although protection in animal

The family Flaviviridae, of which yellow fever virus (YFV) is the prototype, comprises approximately 60 enveloped, positive strand RNAviruses that cause significant public health problems in both temperate and tropical regions of the world (Shope, 1980; Monath, 1986). Although a safe and effective live attenuated vaccine has been available for yellow fever for 50 years (Theiler and Smith, 1937), attempts to produce similar vaccines against other flaviviruses using conventional methods, most notably dengue (Brandt, 1988), have met with limited success. Flavivirus proteins are encoded by a single translational open reading frame (ORF) arranged as follows: 5’-C-prM-E-NSl -NS2a-NS2b-NS3-NS4a-NS4b-NS5-3’. The genes encoding the virion structural proteins (capsid, premembrane, and envelope) are positioned at the 5’ end of the genome followed by at least seven nonstructural proteins, including the nonstructural glycoprotein NSI (Chambers et a/., 1990). Protection from lethal viral infection has been achieved with passively administered monoclonal antibodies (MAbs) specific for each of the three flavivirus glycoproteins (prM, E, and NSl) (Heinz et a/., 1983; Mathews and Roehrig, 1984; Schlesinger et al., 1985; ’ To whom correspondence dressed. 0042-6822/92

and reprint requests

53.00

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form rescwcd.

should be ad-

290

YFV-VACCINIA

model systems has not always correlated with the production of neutralizing antibodies. We have previously described a recombinant vaccinia virus which correctly expressed JEV prM, E, and NSl proteins, and which induced the synthesis of extracellular particles that behaved like empty viral envelopes and protected mice from a lethal JEV challenge (Mason et a/., 1991). In the current study, we have developed vaccinia recombinants expressing portions of the YFV ORF extending from C to NS2B in order to further define the requirements for effective flavivirus vaccines. MATERIALS

AND METHODS

Cell lines and virus strains A host range mutant of vaccinia virus (WR strain) VP293 (Perkus et al., 1989) was used to generate all recombinants (see below). vP457 was derived from VP293 by the restoration of the Kl L host range gene (Perkus eta/,, 1989). All vaccinia virus stocks were produced in eitherVER0 (ATCC CCL 81) or MRC-5 (ATCC CCL 171) cells in Eagle’s MEM supplemented with 5-l 0% newborn calf serum (NCS) (Flow Laboratories, McLean, VI.). Biosynthetic studies were performed using VERO cells grown at 37” in MEM supplemented with 5010NCS or HeLa (ATCC CCL 2) cells grown in MEM supplemented with 10% fetal bovine serum and nonessential amino acids. The YFV-17D stock used in all in vitro and in viva protection experiments was a concentrated fraction obtained by ultracentrifugation of culture fluid prepared from C6/36 cells (Igarashi, 1978) infected with a passage 2 suckling mouse brain suspension of the 17D strain of YFV. Animal challenge experiments were performed using a passage 55 suckling mouse brain suspension of the French neurotropic strain of YFV. Cloning of YF genes into a vaccinia plasmid

virus donor

Restriction enzymes were obtained from Bethesda Research Laboratories (Gaithersburg, MD), New England Biolabs (Beverly, MA) or Boehringer-Mannheim Corp. (Indianapolis, IN). T4 DNA ligase was obtained from New England Biolabs. Standard recombinant DNA techniques were used (Maniatis et a/., 1982) with minor modifications for cloning, screening, and plasmid purification. Nucleic acid sequences were confirmed using standard dideoxy chain-termination reactions (Sanger et a/., 1977) on alkaline-denatured double-stranded plasmid templates. Oligonucleotides were synthesized using standard chemistries (Biosearch 8700, San Rafael, CA; Applied Biosystems

291

VIRUS PROTECTION

380B, Foster City, CA). The YF 17D cDNA clones (clone 10 III and clone 28 Ill) were previously described (Rice et al., 1988), and all nucleotide coordinates are derived from the sequence data presented in Rice eta/. (1985). YFV cDNA was inserted into the vaccinia virus donor plasmid pHES4. Plasmid pHES4 contains the vaccinia Kl L host range gene, the early/late vaccinia virus H6 promoter, unique multicloning restriction sites, translation stop codons, and an early transcription termination signal (Perkus eta/., 1989). Plasmid YF18 contains the putative 17 aa signal sequence for NSl , NSl , and NS2A (nucleotides 2402-4180) with a potential vaccinia virus early transcription termination signal (Yuen and Moss, 1987; TTTTTGT nucleotides 2429-2435) mutated. Plasmid YF23 contains the putative 19 aa signal sequence for E, E, NSl, NS2A, and NS2B (nucleotides 917-4569) with potential early transcription termination signals (TTTTlTGT nucleotides 18861893 and TTTTTGT nucleotides 2429-2435) mutated. Plasmid YF19 contains C, prM, E, NSl , and NS2A (nucleotides 1 19-4180) with potential early transcription termination signals (TTTTTCT nucleotides 263-269, TTTfTGT nucleotides 269-275, IGT nucleotides 1886-1893, and mGT nucleotides 24292435) mutated. Plasmid YF47 contains the putative 21 aa signal sequence for prM, prM, E, NSl, and NS2A (nucleotides 419-4180) with potential early transcription termination signals (TTTTTTGT nucleotides 18861893 and llTlTGT nucleotides 242992435) mutated. Construction

of vaccinia

recombinants

Procedures for transfection of recombinant donor plasmids into tissue culture cells infected with a rescuing vaccinia virus and identification of recombinants by host range selection and in situ hybridization on nitrocellulose filters have been described (Perkus et al,, 1989). YF18, YF23, YFl9, and YF47 were transfected into vP293-infected cells to generate the vaccinia recombinants vP725, vP729, vP766, and vP869, respectively (Fig. 1). In vitro virus infection

and radiolabeling

Hela cell monolayers prepared in 35-mm diameter dishes were infected with vaccinia viruses (m.o.i. of 2) or YFV-17D (m.o.i. of 4). At 16 hr (vaccinia) or 38 hr (YFV-17D) postinfection, the cells were pulse labeled for 2 hr with medium containing [35S]Met and chased for 6 hr in the presence of excess unlabeled methionine as described by Mason et a/. (1991). Radioimmunoprecipitation electrophoresis

and polyacrylamide

gel

Radiolabeled cell lysate and culture fluid were prepared and the viral proteins were immunoprecipitated

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PINCUS ET AL.

and resolved by SDS-containing polyacrylamide electrophoresis (SDS-PAGE) as described by Mason et al. (1991) using the flavivirus cross-reactive anti-E (Dl4G2; Henchal, et al., 1985) or anti-NSl (D2-7El 1; Mason et al., 1990) MAbs. Animal protection

experiments

to

Pooled sera were tested for their ability to precipitate YFV-17D proteins from a detergent-treated cell lysate obtained from [35S]Met labeled YFV-1 -/D-infected cells as described by Mason et al. (199 1). Hemagglutination and hemagglutination inhibition (HAI) tests and neutralization (NEUT) tests were performed using YFV-17D essentially as described by Mason eta/. (1991) with the exception that fresh human sera was not used in the NEUT test. RESULTS Construction of recombinant vaccinia viruses. Four different vaccinia virus recombinants that expressed portions of the YFV coding region extending from C through NS2B were constructed utilizing a host range selection system (Perkus et a/., 1989). The YFV cDNA sequences contained in these recombinants are shown in Fig. 1. In all four recombinant viruses the sense strand of YFV cDNA was positioned behind the vaccinia virus early/late H6 promoter, and translation was expected to be initiated from Met codons located at the 5’ ends of the viral cDNA sequences (Fig. 1). Recombinant vP725 encodes the putative 17-aa signal sequence preceding the N terminus of the nonstructural protein NSl and the nonstructural proteins NSl and NS2A. Recombinant VP729 encodes the putative 19-aa signal sequence preceding the N terminus

prM I

vP766

VP869

VP729

Groups of 20 3-week-old outbred Swiss mice were immunized by intraperitoneal (i.p.) injection with lo7 PFU of vaccinia virus or 1O6 PFU of YFV-17D, and 3 weeks later sera were collected from selected mice. One half of the mice in each group were then either reinoculated with the recombinant virus orYFV-17D, or challenged by intracranial (i.c.) injection with a 1: 1O5 dilution of brain homogenate prepared from suckling mice infected with the French neurotropicYFV(approximately 100 LD,,). Three weeks later, the boosted animals were bled and then challenged with the French neurotropic strain of YFV. Following challenge, mice were observed daily for 3 weeks. Lethal-dose titrations were performed in each challenge experiment using litter-mates. Evaluation of YFV-specific immune responses recombinant vaccinia virus inoculation

C

vP725

E

I

I

NSl

I

NS2B

NS2A

I

11111111111111111 ’

I

I

I

I

I

I

FIG. 1. Map of the YFV coding regions inserted in the recombinant vaccinia viruses. The NSl and NS2A regions in VP869 are depicted as a broken line since this recombinant is missing a base within the NSl coding region.

of E, as well as E, NSl , NS2A, and NS2B. Recombinant vP766 encodes C, prM, E, NSl, and NS2A. Recombinant VP869 encodes the putative 21-aa signal sequence preceding the N terminus of the structural protein precursor prM, as well as prM, E, NSl, and NS2A. However, nucleotide 2962 (within NSl) was inadvertently deleted in the donor plasmid (YF47) used to derive this recombinant as determined by subsequent DNA sequence analysis. In all four recombinants potential vaccinia virus early transcription termination signals (TrKTNT) in C (TKlXTTTTTGT nucleotides 263-275) and in E (TITTTTGT nucleotides 1886-l 893 and TTTTTGT nucleotides 2429-2435) were modified without altering the aa sequence. This change was made in an attempt to increase the level of expression since this sequence has been shown to increase transcription termination in in vitro transcription assays (Yuen and Moss, 1987) and in viva (Earl et al,, 1990). E expression by recombinant vaccinia virus. The data from the pulse-chase experiment depicted in Fig. 2 demonstrate that HeLa cells infected with VP869 produce a protein antigenically related to E and equivalent in size to E synthesized in YFV-17D-infected cells. Under these conditions of pulse-labeling, very little E protein was detected in cells infected with vP766 or vP729. The extracellularfluid from vP869- or YFV-17Dinfected cells contained an equivalent sized E protein, whereas no E protein was detected in the culture fluid harvested from vP766- or vP729-infected cells. Vero cells were infected with the vaccinia recombinants (m.o.i. of 10) and labeled for 8 hr with [35SlMet. The cell monolayer or culture fluid was harvested and immunoprecipitated with a MAb to E. Under these conditions, vP766- and vP729-infected cultures clearly produced a protein equivalent in size to E produced by vP869-infected cultures (data not shown). The low level of expression of E in vP766 or vP729-infected

YFV-VACCINIA

17D 869 729 725 ---MFMFMFMFMFki-F

766

457

FIG. 2. Comparison of the E protein produced by YFV infection or infection with the recombinant vaccinia viruses. HeLa cells were Infected with YFV-17D or recombinant vaccinia viruses, labeled for 2 hr with [35S]Met, and chased for 6 hr. Equal portions of the cell monolayer (M) or culture fluid (F) prepared from each layer were immunoprecipitated with a MAb specific for E and then subjected to SDS-PAGE analysis.

cells under pulse-chase conditions may reflect a difference in its stability due to the presence of C in vP766 and lack of prM in vP729. The extracellular fluid harvested from HeLa cells infected with VP869 contained a hemagglutinating activity that was not detectable in the culture fluid of cells infected with vP457, vP725, vP729, orvP766 (Table 1). This hemagglutinin had a titer and a pH optimum similar to that of the extracellular hemagglutinin produced by YFV-17D-infected cells. Sucrose gradient analyses showed that this extracellular HA sediments as a particle and comigrates with the extracellular HA from YFV17D-infected cells (data not shown). NS 1 expression by recombinant vaccinia virus. The data from the pulse-chase experiment depicted in Fig. 3 demonstrate that varying amounts of a protein antigenitally related to NSl and equivalent in size to YFV17D NSl were synthesized in HeLa cells infected with vP725, vP729, and vP766. The complete sensitivity of the vP725 cell-associated NSl to endo H and PNGase

TABLE 1 EXTRACELLULARHA PRODUCEDBY RECOMBINANT VACCINIA-INFECTEDHELA CELLS Recombinant VP457 vP725 VP729 vP766 VP869

virus

HA titer of culture fluid <1:2 <1:2 <1:2 <1:2 1:64

VIRUS PROTECTION

293

17D 869 729 -M F M F M

725 766 M MT

457 M

FIG. 3. Comparison of the NSl protein produced by YFV infection or infection with the recombinant vaccinia viruses. HeLa cells were infected with YFV-17D or recombinant vaccinia viruses, labeled for 2 hr with [%]Met, and chased for 6 hr. Equal fractions of the cell monolayer (M) or culture fluid (F) prepared from each cell layer were immunoprecipitated with a MAb specific for NSl and then subjected to SDS-PAGE analysis.

F indicated that it was glycosylated; specifically, it contained immature endo H (sensitive) N-linked glycans (data not shown). As expected, VP869 did not express NSl due to a spontaneous nucleotide deletion in the NSl region of the donor plasmid used to derive this recombinant. Varying amounts of NSl produced by YFV-17D, vP725, and VP729 were released into the culture fluid of infected HeLa cells in a higher molecular weight glycosylated form, and the extracellular NSl from YFV17D and vP725 were sensitive to PNGase F digestion (data not shown) consistent with previous studies of the synthesis and secretion of YFV NSl (Post et al., 1990; Despres eta/., 1991). The culture fluid from cells infected with vP766 did not contain detectable NSl.

Recombinant vaccinia viruses induced immune responses to YFVantigens. We tested the recombinant vaccinia viruses for their ability to protect outbred mice from lethal i.c. challenge with the French neurotropic strain of YFV. In our challenge experiments we examined the effect of either a primary or a primary plus booster inoculation on the levels of protection. Serological responses in a subset of the vaccinated animals were evaluated. Prechallenge sera pooled from selected animals in each group were tested for their ability to immunoprecipitate radiolabeled E and NSl The results of these studies (Fig. 4) demonstrated that (1) onlyvP869- and YFV-1 -/D-immunized mice responded to E; (2) mice immunized with YFV-17D, vP766, vP729, or vP725 responded to NSl; and (3) all immune responses were increased by a second inoculation with the recombinant virus. Analysis of the NEUT and HAI data from the sera collected from these animals confirmed the results of the immunoprecipitation analysis,

294

PINCUS ET AL. A IMMUNE MAb

YFV-17D

E NSI

1

1

VP889 2

B

RESPONSE

IMMUNE

VP729

1 I 1 I 2

1

1

vP725 2

1

1

RESPONSE VP457

vP766 2

1

1

2

1

1

2

-

E NSl dye FIG. 4. Analysis of the YFV-specific reactivity of prechallenge sera from mice vaccinated with the recombinant vaccinia viruses. Sera collected from a subset of the animals used in the protection experiments (Tables 2 and 3) were pooled and aliquots were tested for their ability to immunoprecipitate radiolabeled proteins from cell lysates of YFV-infected cells. (A) The position of the E and NSl proteins precipitated with MAbs and the radioimmunoprecipitations obtained from the pooled sera of animals vaccinated once (1) or twice (2) with YFV-17D, VP869 or vP729. (B) The reactivity of sera obtained from animals vaccinated with vP725, vP766 or vP457 or from uninoculated mice (-).

showing that only vP869- and YFV-17D-immunized mice had high levels of NEUT and HAI antibodies (Table 2). Vaccination with VP869 provided protection from lethal YFV infection. Mice immunized once or twice with VP869 or YFV-17D were protected from French neurotropic virus challenge. Levels of protection achieved by immunization with VP869 were equivalent to those achieved by immunization with YFV-17D (Fisher Exact Test P < 0.05; Table 3). Mice immunized with vP766, vP729, orvP725 expressing NSl were not

TABLE 2 NEUT AND HAI ANTIBODYTITERSINTHEPRECHALLENGESERA One inoculation Immunizing virusa vP457 vP457 vP725 vP725 VP729 VP729 vP766 vP766 VP869 VP869 YF17D YF17D

Group Group Group Group Group Group Group Group Group Group Group Group

1 2 1 2 1 2 1 2 1 2 1 2

NEUT tite?

HAI titerC



Two inoculations NEUT tite?

HAI titerC

significantly protected from French neurotropic virus challenge when compared to mice inoculated with the controlvacciniavirusvP457, even though these recombinants elicited immune responses to the NSl protein. DISCUSSION We constructed four different YFV-vaccinia recombinants to further understand the requirements for effective flavivirus vaccines. Evaluation of these recombinants in a mouse challenge system showed that one of these viruses, vP869, induced high levels of neutralizing and hemagglutination-inhibiting antibodies in inoculated mice and was capable of protecting mice from a challenge with the French neurotropic YFV. This recombinant vaccinia virus produced a particulate extracellular hemagglutinin similar to the extracellular SHAlike particles produced by JEV-vaccinia recombinants described previously (Mason et a/., 1991).

TABLE 3

1:lO

<1:10

<1:10

<1:10

<1:10

<1:10

Immunizing virus

<1:10

<1:10

VP457 vP725 VP729 vP766 VP869 YF17D

PROTECTIONOF MICEFROM FATALYFV ENCEPHALITIS

1:320 >1:2560

1:320 1:1280

aVirus used for immunization: Group 1 indicates animals challenged 3 weeks following a single inoculation and Group 2 indicates animals challenged following two inoculations (see Materials and Methods). * Serum dilution yielding 90% reduction in plaque number. c Serum dilution.

Survival afte? one inoculation O/16 o/14 O/16 o/14 8115 10113

Survival afte? two inoculations l/14 2116 2113 o/14 15/16 16/16

a Live animals/total for each group; 220 LD,, challenge delivered to 6-week-old mice, 3 weeks following a single inoculation. b Live animals/total for each group; 36 LD,, challenge delivered to g-week-old mice, 6 weeks following the first inoculation and 3 weeks following the second inoculation with the same virus and dose.

YFV-VACCINIA

The four recombinant viruses described in this manuscript contain portions of the YFV ORF that encode the capsid protein C, the precursor to the structural protein M, the structural protein E, and nonstructural proteins NSl, NS2A, and NS2B. vP766-, vP869-, and vP729-infected cell cultures synthesized E protein, although only low levels of expression were observed in vP766- and vP729-infected cells, possibly reflecting a difference in stability due to the presence of C (vP766) and the absence of prM (vP729). vP869, but not vP766 and vP729, synthesized an extracellular hemagglutinin, suggesting that the presence of prM and the absence of C are required for production of this extracellular HA, as we have proposed for JEV-vaccinia recombinants (Mason eS al., 1991; Konishi et al., 1991). Since VP869 does not express NSl, NSl is not required for formation of the extracellular HA in agreement with our recent results on the expression of an extracellular HA byvaccinia-JEV recombinants (Konishi et al., 1991). vP766-, vP725-, and vP729-infected cells contained varying amounts of an NSl protein equivalent in size to NSl in YFV-17D-infected cells. vP725- and vP729-infected cells secreted variable amounts of a NSl protein equivalent in size to NSl secreted by YFV-17D-infected cells. However, cells infected with the recombinant vP766 which contained the YFV ORF from C through NS2A failed to secrete detectable NSl. Although we have not formally demonstrated that vP766-infected cells contain any form(s) of the C protein, we feel that the C protein in the absence of other viral components (including RNA and proteinase) may effect the release of both E and NSl, a result consistent with vaccinia-JEV recombinants (Konishi et a/., 1991). Recombinants vP766, vP729, and VP725 induced an immune response to NSl, but failed to protect mice against the French neurotropic virus challenge. This result contrasts with the studies of Putnak and Schlesinger (1990) showing that a vaccinia recombinant expressing YFV-17D NSl could partially protect mice against YFV-17D challenge. Gould et al. (1986) previously demonstrated that the ability of YFV NSl monoclonal antibodies to protect against infection depended on the degree of neurovirulence of the challenge virus for mice. Additional variability in the ability of NSl to protect against infection has been reported to depend on the flavivirus studied. Vaccinia virus expressing dengue-4 NSl conferred solid immunity (Falgout et a/., 1989) whereas vaccinia recombinants expressing JEV NSl provided only low levels of protection (Konishi et al., 1991). Several reports from other laboratories have documented the ability of flavivirus-vaccinia recombinants

295

VIRUS PROTECTION

to protect mice from lethal flavivirus challenge (see Introduction). We have focused on the production of extracellular particles by JEV-vaccinia constructs as the criteria for producing effective vaccines (Mason et al., 1991; Konishi eta/., 1991). The inability of vP766 to protect mice against the French neurotropic virus challenge contrasts with the results described for Dengue 4-vaccinia (Zhao et a/., 1987; Bray et al., 1989) and JEV-vaccinia constructs (Konishi et al., 1991) expressing the same region of the genome. Also, the lack of protection by VP729 contrasts with the results described with an analogous JEV-vaccinia construct (Mason et al., 1991). This apparent inconsistency in the immunogenicity of the cellassociated E proteins could reflect differences in the ability of these proteins to be presented to the immune system in viva, possibly due to differences in their stability. It is clear, however, that constructs expressing prM and E and producing an extracellular hemagglutinin can protect mice against YFV or JEV challenge. These types of constructs seem excellent candidates for effective flavivirus vaccines.

ACKNOWLEDGMENTS Ascitic fluids produced from hybridomas Dl-4G2 and D2-7El 1 were supplied by Dr. Mary Kay Gentry (WRAIR, Washington, DC). This work was supported by grants from the National Institutes of Health, Al 10987-l 7, the U.S. Army Medical Research and Development Command, DAMDl7-87-G-7005, and the National Science Foundation, DMB 8515345. E.K. was supported by the Department of Medical Zoology, Kobe University School of Medicine, Kobe, Japan. B.F. was supported in part by a scholarship from the Conselho National de Desenvolvimento Cientifico e Tecnologico-CNPq of the Ministry for Science and Technology of Brazil. We thank Dr. E. Norton, C. Molnar, S. Goebel, J. Smith, H. Martinez, J. Winslow, and K. Catlin for their contributions to various aspects of this work.

REFERENCES BRANDT, W. E. (1988). Current approaches to the development of dengue vaccines and related aspects of the molecular biology of flaviviruses. 1. Iniect. Dis. 157, 1 105-l 1 1 1, BRAY, M., ZHAO, B., MARKOFF, L., ECKELS,K. H., CHANOCK, R. M., and LAI, C.-J. (1989). Mice immunized with recombinant vaccinia virus expressing dengue 4 virus structural proteins with or without nonstructural protein NSl are protected against fatal dengue virus encephalitis. /. Viral. 63, 2853-2856. CANE, P. A., and GOULD, E. A. (1988). Reduction of yellow fevervirus mouse neurovirulence by immunization with a bacterially synthesized non-structural protein (NSl) fragment. /. Gen. Virol. 69, 1241-1246. CHAMBERS, T. J., HAHN, C. S., GALLER, R., and RICE, C. M. (1990). Flavivirus genome organization, expression, and replication. Annu. Rev. Microbial. 44, 649-688. DESPRES,P., GIRARD, M., and BOULOY, M. (1991). Characterization of yellow fever proteins E and NSl expressed in Vero and Spodoptera frugiperda cells. /. Gen. Viral. 72, 133 1-l 342. DEUBEL, V., BORDIER, M., MEGERT, F., GENTRY, M. K., SCHLESINGER, J. J., and GIRARD, M. (1991). Processing, secretion, and immunore-

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