Recombinant vaccinia viruses co-expressing dengue-1 glycoproteins prM and E induce neutralizing antibodies in mice

Recombinant vaccinia viruses co-expressing dengue-1 glycoproteins prM and E induce neutralizing antibodies in mice Benedito A.L. Fonseca*, Steven Pincus t, Robert E. Shope*, Enzo Paoletti t and Peter W. Mason * ~ Four recombinant vaccinia viruses expressing different portions of the dengue type 1 virus (DEN-l) genome (C-prM-E-NS1-NS2A-NS2B; prM-E; prM-E-NS1-NS2A-NS2B; or NS1-NS2A) were constructed in order to establish the most immunogenic configuration of DEN-1 proteins. Both recombinants producing prM and E in the absence of C induced the synthesis of extracellular forms of E in vitro. Mice inoculated with these two recombinants produced DEN-1 neutralizing (NEUT) and haemagglutination inhibiting (HAI) antibodies. The other two recombinant vaccinia viruses, which did not induce the production of extracellular forms of E, did not induce E-specific immune responses. These results support our previous studies on the design of flavivirus-vac¢inia vaccine candidates by showing the importance of co-expressing prM and E in order to induce the synthesis of extracellular E and to elicit NEUT and HA1 antibodies. Keywords:Dengue; vaccinia; recombinant; immunogenicity

Dengue viruses occur as four antigenically related but distinct serotypes that are biologically transmitted from infected to susceptible humans mainly by Aedes aegypti mosquitoes. These viruses are among the most important human pathogens of the family Flaviviridae ~. Dengue virus infections induce diseases ranging from dengue fever to dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS). Dengue fever is a self-limited febrile illness, whereas DHF/DSS is a life-threatening disease more commonly observed in secondary infections by a serotype that is different from the one that caused the primary infection. It has been hypothesized that antibodies remaining from a primary infection bind virus during a secondary infection, leading to the appearance of circulating immune complexes without aborting infection. Furthermore, since antibody-complexed virus can infect mononuclear cells expressing the immunoglobulin (Fc) receptor in vitro2, these antibodies may actually enhance lnfectton m vwo. This enhancement hypothesis L3 and other possible cellular immune responses 4,5 suggest that the immune system plays an important role in DHF/DSS pathogenesis. *Yale Arbovirus Research Unit, Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, CT 06510, USA. t Vi rogenetics Corporation, 465 Jordan Road, Rensselaer Technology Park, Troy, NY 12180, USA. t Plum Island Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Greenport, NY 11944, USA. °°To whom correspondence should be addressed. (Received 23 April 1993; revised 8 June 1993; accepted 8 June 1993) 0264-410X/94/03/0279-07 © 1994 Butterworth-Heinemann Ltd

There is no specific treatment available for dengue disease and the control of epidemic transmission is based only on management of the vector population. Since 100 million new cases result each year from dengue outbreaks, a vaccine is needed 1. Attenuated vaccine candidates have been reported for three of the four DEN serotypes 6. Even with the development of a tetravalent vaccine, these vaccines may be subject to several important theoretical drawbacks, including reversion to virulence and competition among the four vaccine viruses for infection of susceptible host cells. Developments in the understanding of the molecular biology of flaviviruses 7 have provided new approaches fo~ the production of dengue vaccines. Among the flavivirus-encoded proteins, prM, E and NS1 (see Figure1) have been identified as protective immunogens by passive protection experiments with monoclonal antibodies (mAbs) a-16. Based on findings that NS1 mAbs 1° or immunization with NS117'~8 could protect against infection, work on the production of NSl-based vaccine candidates has been pursued in several laboratories since enhancement of infection was linked to structural protein antibodies (see above, Ref. 1). Several different recombinant DNA strategies have been employed to generate candidate flavivirus vaccines. The most successful strategies to date have included recombinant baculovirus-generated proteins ~9-24, and live recombinant vaccinia viruses 25-38. We have previously shown that vaccinia recombinants expressing Japanese encephalitis virus (JEV) NS 1 protein could provide partial protection from infection 35,

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DEN-1 GENOME 10,200 base OPEN READING FRAME

5'-~

I

t-3' NONSTRUCTURAL

STRUCTURAL

MAJOR PROTEOLYTIC PRODUCTS

h,\\\\\\\\\Nii:i:l I~\\\\\\~1

~ anch.C

prM

c

E

NS1

ns2a,b

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NS3

II

ns4a,b

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~, cleavage sites [~ glycoproteins I N-terminal signal peptides C-terminal membrane anchor/signal peptides

M

RECOMBINANT VACCINIA VIRUS GENOMES

vP833

I

C

[ prM

I

E

vP962

I

prM

I

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I

vP1027

1

prM

[

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I

vP841

1

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I NS2B I

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J

Figure 1 Schematic representation of DEN-1 virus genome and the portions of DEN-10RF inserted into the recombinant vaccinia viruses. Filled rectangles represent N-terminal signal peptides. NS1-2B of vP833 are shaded, as a fortuitous deletion of nucleotide 2894 (within NS1) in this construct prevented proper translation of these three proteins

consistent with the results of Falgout et al. 29 for DEN-4. However, in our yellow fever (YF) challenge model, we failed to observe protection with a vaccinia recombinant expressing YF/NS138, in contrast to the results of Putnak and Schlesinger 31. In these same studies, we reported that recombinant vaccinia viruses that correctly expressed the prM and E proteins from JEV or YF induced the synthesis of extracellular particles that behaved like empty viral envelopes and protected mice from a lethal challenge 33'35'36'38. Following the same line of research, we have constructed recombinant vaccinia viruses that express the DEN-1 prM and E genes and tested their ability to induce the synthesis of extracellular forms of E protein and elicit an immune response to DEN-1 in mice. MATERIALS AND METHODS Cell lines and virus strains

A thymidine kinase mutant of the Copenhagen strain of vaccinia virus vP41039 was used to generate recombinants vP833 and vP841, vP452, which was derived from vP410 by deletion of the HA gene4°, was used to generate vP962 and vP1027. Figure 1 provides a schematic representation of the DEN-1 sequences incorporated into each recombinant virus. Vaccinia virus stocks were produced in Vero (ATCC CCL81) cells in Eagle's minimal essential medium (MEM) plus 10% heat-inactivated fetal bovine serum (FBS). Biosynthetic studies were performed using HeLa (ATCC CCL2) cells grown at 37°C in MEM supplemented with 10% FBS, antibiotics and non-essential amino acids. The strain of DEN-I used in in vitro experiments was the Nauru Island

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strain (Fetal Rhesus Lung p8, C6/36 p2, Veto pl4X). Mice were immunized with DEN-1 Hawaii (suckling mouse brain p2242). Cloning of DEN-1 genes into plasmids from a vaccinia virus donor

Restriction enzymes and T4 DNA ligase were obtained from BRL (Bethesda, MD), New England Biolabs (Beverly, MA) or Boehringer Mannheim Biochemicals (Indianapolis, IN). Standard recombinant DNA techniques were used 43 with minor modifications for cloning, screening and plasmid purification. Nucleic acid sequences were confirmed using standard dideoxy chain-termination reactions on alkaline-denatured doublestranded plasmid templates 44. Oligonucleotides were synthesized using standard chemistries (Biosearch 8700, San Rafael, CA; Applied Biosystems 380B, Foster City, CA). Plasmids containing eDNA encoding C, prM, E and NS1 from the Nauru Island strain of DEN-1 have beel5 described 45. Additional sequences from the NS2A and NS2B regions of the genome have been deposited with GenBank. DEN-1 eDNA was inserted into the vaccinia virus donor plasmid pTP15 which contains the early/late vaccinia virus H6 promoter, a polylinker region and sequences flanking the vaccinia HA gene4°. Plasmid Den27 contains C, prM, E, NS1, NS2A and NS2B (nucleotides 68-4492); however, nucleotide 2894 (within NS1) was deleted during construction of this plasmid. Plasmid Den34 contains the putative 20 amino acid (aa) signal sequence for prM, prM, E, NS1, NS2A and NS2B (nucleotides 350-4492). Plasmid Den38 encodes the putative 21 aa signal sequence for prM, prM and E (nucleotides

Immunogenic DEN-1 vaccinia recombinants: B.A.L. Fonseca et al.

350-2392). Plasmid Den30 encodes a synthetic ATG, the putative 15 aa signal sequence for NS1, NS1 and NS2A (nucleotides 2348-4102). In all donor plasmids, potential vaccinia virus early transcription termination signals (T5NT 47) within prM (nucleotides 822-828 TTTTTCT) and within NS1 (nucleotides 2448-2454 TTTTTGT) were mutated (to TATTTCT or TATTTAT) without changing the aa sequences. These changes were made in an attempt to maximize the level of expression, since this sequence has been shown to increase transcription termination in in vitro transcription assays46 and to cause transcription termination resulting in apparent decreased expression and immunogenicity of a foreign gene by a recombinant vaccinia virus in vivo 47.

Construction of vaccinia virus recombinants Procedures for transfection of recombinant donor plasmids into tissue culture cells infected with a rescuing vaccinia virus and identification of recombinants by in situ hybridization on nitrocellulose filters have been described 39'48. Plasmids Den27 and Den30 were transfected into vP410-infected cells to generate the vaccinia recombinants vP833 and vP841. Plasmids Den34 and Den38 were transfected into vP452-infected cells to generate recombinants vP962 and vP1027, respectively. In vitro infection, radiolabelling, radioimmunoprecipitation and polyacrylamide gel electrophoresis

HeLa cell monolayers prepared in 35 mm diameter dishes were infected with vaccinia viruses (multiplicity of infection (m.o.i.)=2) or DEN-1 (Nauru Island strain; m.o.i.=5). At 16h (vaccinia) or 72h (DEN-l) postinfection, the cells were pulse-labelled for 2 h with medium containing [aSS]Met and chased for 6 h in the presence of excess unlabelled Met as described by Mason et al. 33. Radiolabelled cell lysates and culture fluids were prepared as described by Mason 49. The viral proteins were immunoprecipitated by two mAbs, D1-4G25° and D2-7El151, specific for the glycoproteins E and NS1, respectively, and resolving by SDS-containing polyacrylamide gel electrophoresis (SDS-PAGE52).

Animal experiments Groups of ten 3-week-old outbred Swiss mice were immunized by intraperitoneal (i.p.) injection with l0 Tp.f.u, of the vaccinia viruses or DEN-1 (Hawaii strain). Three weeks later, sera were collected from selected mice, and the mice were reinoculated with the respective recombinant viruses or DEN-l, (Hawaii strain). Three weeks later, the boosted animals were rebled. Mice immunized once were given an i.p. injection of vaccinia virus or DEN-1 at 6 weeks of age and sera were collected from selected mice 3 weeks later. Pooled sera were tested for their ability to precipitate DEN-1 proteins from detergent-treated culture fluids obtained from [35 S] Met-labelled DEN- 1-infected cells 3 3. Haemagglutination inhibition (HA1) tests 53 were performed by slight modifications of existing protocols. The neutralization (NEUT) tests 54 were performed in Vero cells, using paired 1.4cm diameter wells for each virus/antibody dilution, and values reported are serum dilutions yielding a 95% reduction in plaque number.

RESULTS

Structures of the recombinant vaecinia viruses Four vaccinia recombinants were constructed that contained portions of the DEN-1 coding region extending from C through NS2B inserted in the vaccinia HA locus. In all recombinant viruses, the sense strand of the DEN-1 eDNA was cloned behind the early/late H6 promoter, and translation was expected to be initiated from naturally occurring DEN-1 Met codons located at the 5' ends of the viral eDNA sequences or a synthetic ATG provided at the 5' end of the coding region of the NS1 gene inserted into vP841 (Figure 1).

E is properly processed when expressed by recombinant vaccinia viruses Biochemical and immunological analyses indicated that the DEN-1 E protein expressed in HeLa cells infected by vP962 and vP1027 was identical to E produced by DEN-l-infected cells. The cell-associated forms of E precipitated by mAb D1-4G2 were the same size as E synthesized in DEN-l-infected cells (Figure 2). E was also released from cells infected with vP1027, and prolonged exposure of the gel used to prepare the autoradiogram (Figure 2) showed that E was also released from vP962-infected cells, but in much smaller amounts (results not shown). E protein released from cells infected with vP1027 appeared to be in subviral particles similar to those derived from JEV-vaccinia recombinants 36, since the vP1027-encoded E cosedimented with the recombinant JEV E proteincontaining particles in sucrose density gradients (results not shown). Interestingly, under pulse-chase conditions, only a small amount of a protein that co-migrated with E in SDS gels was precipitated from lysates of cells infected with vP833 (which encodes C, prM and E). Under conditions of continuous labelling (8 h), cells infected with this recombinant clearly produced a protein of the correct size which was recognized by an E-specific mAb (data not shown). The poor expression of E by vP833 might reflect a difference in its stability due to improper processing when E is co-expressed with C, a result consistent with our previous studies with similar recombinant vaccinia viruses encoding C, prM and E of JEV and YFV 35'38. To further test whether E was properly expressed by vP 1027, we examined its cleavage with endoglycosidases. E proteins from both DEN-1 and vP1027 were identically glycosylated, and the extracellular forms of E contained

Rgure 2 Comparison of E protein expression by HeLa cells infected with the recombinant vaccinia viruses or DEN-1 virus. HeLa cells were infected with the indicated viruses, labelled for 2 h with [=]-Met and chased for 6 h. DEN-1 E proteins present in infected cell monolayers (M) and culture fluids (CF) were immunoprecipitated with mAb specific for E and then subjected to SDS-PAGE analysis

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two N-linked glycans, one immature (endo-H sensitive) and one complex (endo-H resistant, PNGase F sensitive)

with vP1027, vP962, vP841, vP833 or the parental vaccinia virus (vP452) or DEN-1 Hawaii strain. Pooled sera collected from each group after the immunizations were tested for DEN-1 antibody titres. The NEUT and HA1 data for these sera are shown in Table 1. Recombinant viruses vP962 and vP1027 elicited levels of NEUT and HA1 antibodies comparable with those elicited by the DEN-1 virus. On the other hand, vaccinia viruses vP833, vP841 and vP452 did not elicit either NEUT or HA1 antibodies in mice (Table 1). The ability of postimmunization sera to immunoprecipitate authentic DEN-1 antigens was also tested. Sera collected after the first inoculation with DEN-I, vP962 or vP841 were able to immunoprecipitate NS1. Sera from mice inoculated with vP452, vP833 (which contains a deleted copy of the NS1 gene) and vP1027 (which did not contain the NS1 gene) did not precipitate NS 1, consistent with the fact that cells infected with these recombinant viruses in vitro did not produce NS1. None of the pooled sera collected after the first inoculation, including those collected from animals inoculated with DEN-l, was able to immunoprecipitate detectable amounts of E. Following the second immunization, clear reactions with E were obtained with sera from mice immunized with DEN-l, vP962 and vP1027 (Figure 5). The relative ability of these sera to precipitate E agreed with the results of the NEUT and HAI tests, which provide a more quantitative evaluation of immune response to E.

(Figure 3). NS1 is properly processed when expressed by recombinant vaccinia viruses NS1 proteins were expressed in cells infected with the recombinant vaccinia viruses (vP962 and vP841) that contained the complete NS1 and NS2A coding regions (Figure 4). Cells infected with these viruses also produced a secreted form of NS1. The secreted and cell-associated forms of NS1 protein expressed by these two viruses were indistinguishable from the DEN-l-expressed NS1 protein in terms of mobility in SDS-PAGE, and were identically glycosylated as determined by cleavage by endoglycosidases (data not shown), vP833 did not induce the expression of a normal NS1 protein, consistent with the fact that a base was deleted from the NS1 coding region during vP833 donor plasmid construction. However, the precipitation of a protein of higher molecular weight from vP833-infected cells by the NS1 mAb (not shown) suggests that the DEN-1 polyprotein encoded by vP833 was translated in infected cells. A truncated protein reactive with a mAb to NS1 was also detected under conditions of continuous labelling (data not shown).

Recombinant vaccinia viruses induced immune responses to DEN-I antigens In order to test the immunogenicity of the recombinant vaccinia viruses, groups of Swiss mice were inoculated

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Figure 4 Comparison of NSl protein expression by HeLa cells infected with the recombinant vaccinia viruses or DEN-1 virus. HeLa cells were infected with the indicated viruses, and NS1 proteins were radiolabelled and immunoprecipitated with mAb specific for NS1 and then subjected to SDS-PAGE analysis, as described in Figure 2

Figure 3 Endoglycosidase digestion of DEN-1 E protein produced by HeLa cells infected with DEN-1 or vP1027. E proteins were prepared as described in Figure 2, mock-digested (M), or digested with endoglycosidase H (H) or PNGase F (F) and analysed by SDS-PAGE, as described in Methods

Table 1

Immunological response to DEN-1 or recombinant vaccinia viruses Single inoculation a 6 weeks

3 weeks

Double inoculations ~

Viruses

HAl c

PRNT ~

HAl

PRNT

HAl

PRNT

DEN-1 vP1027 vP962 vP841 vP833 vP452

1:20 1:20 <1:20 <1:20 < 1:20 <1:20

1:10 <1:10 <1:10 <1:10 < 1:10 <1:10

1:20 1:20 <1:20 <1:20 < 1:20 <1:20

1:10 1:10 <1:10 <1:10 < 1:10 <1:10

1:40 1:40 1:40 <1:20 < 1:20 <1:20

1:20 1:40 1:40 <1:10 < 1:10 <1:10

aGroups of ten 3-week-old or 6-week-old mice were inoculated i.p. with 10z p.f.u, of DEN-1 (Hawaii) or vaccinia viruses ~Groups of ten 3-week-old mice were inoculated with 10~ p.f.u, of DEN-1 (Hawaii) or vaccinia viruses and reinoculated at 6 weeks of age ~Serum dilution inhibiting haemagglutination as described~1 ~Serum dilution yielding 90% reduction in plaque numbeP 2

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Immunogenic DEN-1 vaccinia recombinants: B.A.L. Fonseca et al.

Figure 5 Analysis of specific reactivity of postimmunization sera to DEN-1 proteins. Mice were immunized with one or two inoculations of the indicated viruses at 3 and/or 6 weeks of age and sera were collected 3 weeks later and used to immunoprecipitate DEN-1 proteins. Proteins precipitated by mAbs specific for E and NS1 are shown in the first two lanes. Low intensity of the E band reflects the small amount of antigen present in the preparation used to prepare this particular autoradiogram

proteins correctly and to induce humoral immune responses to the dengue proteins. Two of the viruses constructed for this study, vP1027 and vP962, induced the synthesis of an E protein that was released from infected cells in vitro. The E protein expressed by vP1027 was indistinguishable from E expressed by DEN-1 based on migration in SDS-PAGE and endoglycosidase studies. Recombinant vP962 also expressed an NS1 protein that appeared to be identical with NS1 expressed by DEN-l-infected cells and by cells infected with vP841 (which only contains the NS1 and NS2A genes). The greater amount of extracellular E produced by cells infected with vP1027 (lacking NS1) compared with vP962 (expressing NS 1) is consistent with our results with similar JEV-vaccinia recombinants 35, and suggests that the NS1 protein could be bound to the structural proteins during some stage of viral assembly. One of the recombinant vaccinia viruses (vP833) did not appear to express any of the dengue proteins correctly. In cells infected with vP833, the E protein was poorly expressed. Further, no NSl-specific polypeptides of proper molecular weight were observed in cells infected by this virus. The poor synthesis of E may reflect problems in stability or processing that arise when the E coding sequences are co-expressed with the C coding sequences in flavivirus-recombinant vaccinia viruses 35.3s,5 5. The absence of NS 1 is consistent with the fact that there was a deletion of nucleotide 2894 (in the NS1 gene) in this recombinant. Analysis of p0stimmunization sera from groups of mice immunized with DEN-1 or the recombinant vaccinia viruses show that a single immunization with viruses that expressed extracellular E (DEN-I, vP962 and vP1027) elicited HA1 and NEUT antibodies. For all these viruses, a boost in antibody levels was observed following a-.second inoculation. Although NEUT tritres observed for the DEN-1 and DEN-1 recombinant viruses were lower than those we have observed for other flaviviruses, it is important to note that NEUT titres elicited by the DEN-l-vaccinia recombinants that expressed~xtracellular E were comparable to those obtained with DEN-1 Hawaii immunization. The levels of NEUT antibodies found in mice inoculated with vaccinia virus in this study are higher than those found by other investigators producing DEN-2 or DEN-4-vaccinia recombinants 25'26'28'34'37. In several of these studies, no detectable NEUT antibodies were produced 25'26'37, but in the case of DEN-4, protection from challenge was nonetheless observed 25,3~. We have been unable to

develop a reliable and consistent challenge system for DEN-1 where protection can be seen following immunization with DEN-1 virus. In unpublished studies, mice inoculated with DEN-l, the parental vaccinia virus, or recombinant vaccinia viruses were challenged with the virulent Mochizuki strain of DEN-15v. Surprisingly, none of the animals inoculated with DEN-1 Hawaii was protected, despite the presence of NEUT antibodies in these animals. Among those mice receiving two inoculations of recombinant vaccinia viruses, two of the recombinant vaccinia viruses (vP962 and vP1027) caused a slight delay in the onset of mortality compared with DEN-1 virus and provided mice with some protection; however, these data are not statistically significant. The studies on DEN-1 recombinants reported here support the value of flavivirus-vaccinia recombinants as vaccine candidates, which has been clearly demonstrated with murine studies using other flaviviruses 28'29'31-35'38. We are continuing these studies with the other dengue serotypes where better murine models exist, but ultimately all of these recombinants will need to be tested in primates. ACKNOWLEDGEMENTS Ascitic fluids produced from hybridomas D1-4G2 and D1-7Ell were supplied by Dr Mary Kay Gentry (WRAIR, Washington, DC). This work was supported by grants from the National Institutes of Health, AI 10987-17, the US Army Medical Research and Development Command, DAMD17-90-Z-0020, the World Health Organization, and the National Science Foundation, DMB 8515345. B.A.L.F. was supported in part by a scholarship from the Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico (CNPq) of the Ministry for Science and Technology of Brazil. The authors are grateful to Dr E. Konishi for many helpful suggestions on various aspects of this work. REFERENCES 1 Halstead, S.B. Pathogenesis of dengue: challenges to molecular biology. Science 1988, 239, 476-481 2 Halstead, S.B. and O'Rourke, E.J. Dengue viruses and mononuclear phagocytes. I. Infection enhancement by non-neutralizing antibody. J. Exp. Med. 1977, 1418,210-217 3 Halstead, S.B. The pathogenesis of dengue: molecular epidemiology in infectious disease. Am. J. Epidemiol. 1981, 114, 632~M8 4 Kurane, I., Innis, B.L., Nisalak, A., Hoke, C., Nimmannitya, S., Meager, A. and Ennis, F.A. Human T cell responses to dengue virus antigens. Proliferative responses and interferon gamma production. J. Clin. Invest. 1989, 8,3, 506-513 5 Kurane, I., Meager, A. and Ennis, F.A. Dengue virus-specific human T cell clones: serotype cross-reactive proliferation, interferon gamma production, and cytotoxic activity. J. Exp. Med. 1989, 170, 763-775 6 Bhamarapravati, N. and Yoksan, S. The clinical trial of trivalent dengue vaccine. Southeast Asian J. Trop. Med. Public Health 1990, 21, 709 7 Chambers, T.J., Hahn, C.S., Galler, R. and Rice, C.M. Flavivirus genome organization, expression, and replication. Annu. Rev. Microbiol. 1990, 44, 649-688 8 Heinz, F.X., Berger, R., Tuma, W. and Kunz, C. A topological and functional model of epitopes on the structural glycoprotein of tick-borne encephalitis virus defined by monoclonal antibodies. Virology 1983, 126, 525-537 9 Mathews, J.H. and Roehrig, J.T. Elucidation of the topography and determination of the protective epitopes on the E glycoprotein of Saint Louis encephalitis virus by passive transfer with monoclonal antibodies. J. Immunol. 1984, 132, 1533-1537

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10 Schlesinger, J.J., Brandriss, M.W. and Walsh, E.E. Protection against 17D yellow fever encephalitis in mice by passive transfer of monoclonal antibodies to the nonstructural glycoprotein gp48 and by active immunization with gp48. J. Immuno/. 1985,135, 2805-2809 11 Gould, E.A., Buckley, A., Barrett, A.D.T. and Cammack, N. Neutralizing (54K) and non-neutralizing (54K and 48K) monoclonal antibodies against structural and non-structural yellow fever virus proteins confer immunity in mice. J. Gen. Viro/. 1986, 67, 591-595 12 Kaufman, B.M., Summers, P.L, Dubois, D.R., Cohen, W.H., Gentry, M.K., Timchak, R.L. eta/. Monoclonal antibodies for dengue virus prM glycoprotein protect mice against lethal dengue infection. Am. J. Trop. Med. Hyg. 1989, 41,576-580 13 Kaufman, B.M., Summers, P.L., Dubois, D.R. and Eckels, K.H. Monoclonal antibodies against dengue 2 virus E-glycoprotein protect mice against lethal dengue infection. Am. J. Trop. Med. Hyg. 1987, 36, 427-434 14 Henchal, E,A., Henchal, L.S. and Schlesinger, J.J. Synergistic interactions of anti-NS1 monoclonal antibodies protect passively immunized mice from lethal challenge with dengue 2 virus. J. Gen. Viro/. 1988, 65, 2101-2107 15 Kimura-Kuroda, J. and Yasui, K. Protection of mice against Japanese encephalitis virus by passive administration with monoclonal antibodies. J. Immuno/. 1988, 141, 3606-3610 16 Mason, P.W., Dalrymple, J.M, Gentry, M.K., McCown, J.M., Hoke, C.H., Burke, D.S. eta/. Molecular characterization of a neutralizing domain of the Japanese encephalitis virus structural glycoprotein. J. Gen. Virol. 1989, 76, 2037-2049 17 Schlesinger, J.J., Brandriss, M.W., Cropp, C.B. and Monath, T.P. Protection against yellow fever in monkeys by immunization with yellow fever virus nonstructural protein NSI. J. Viro/. 1986, 60, 1153-1155 18 Schlesinger, J.J., Brandriss, MW. and Walsh, E.E. Protection of mice against dengue 2 virus encephalitis by immunization with the dengue 2 virus non-structural glycoprotein NS1. J. Gen. Viro/. 1987, 65, 853-857 19 Zhang, Y.-M., Hayes, E.P., McCarthy, T.C., Dubois, D.R., Summers, P.L., Eckels, K.H. et al. Immunization of mice with dengue structural proteins and nonstructural protein NS1 expressed by baculovirus recombinant induces resistance to dengue virus encephalitis. J. Viro/. 1988, 62, 3027-3031 20 Matsuura, Y., Miyamoto, M., Sato, T., Morita, C. and Yasui, K. Characterization of Japanese encephalitis virus envelope protein expressed by recombinant baculoviruses. Virology 1989, 173, 674-682 21 McCown, J., Cochran, M., Putnak, R., Feighny, R., Burrous, J., Henchal, E. and Hoke, C. Protection of mice against lethal Japanese encepahalitis with a recombinant baculovirus vaccine. Am. J. Trop. Med. Hyg. 1990, 42, 491-499 22 Deubel, V., Bordier, M., Megret, F., Gentry, M.K., Schlesinger, J.J. and Girard, M. Processing, secretion, and immunoreactivity of carboxy terminally truncated dengue-2 virus envelope proteins expressed in insect cells by recombinant baculoviruses. Virology 1991, 180, 442-447 23 Lai, C.-J., Zhang, Y.-M., Bray, M., Chanock, R.M., Dubois, D.R. and Eckels, K.H. Immunization of monkeys with baculovirus recombinantexpressed dengue envelope and NS1 glycoproteins induces partial resistance to challenge with homotypic dengue virus. In: Vaccines 90: Modern Approaches to New Vaccines including Prevention of AIDS. (Eds Brown, F., Chanock, R.M., Ginsberg, H. and Lerner, R.A.) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1990, pp. 119-124 24 Putnak, R., Feighny, R., Burrous, J., Cochran, M., I-lackett, C., Smith, G. and Hoke, C. Dengue-1 virus envelope glycoprotein gene expressed in recombinant baculovirus elicits virus-neutralizing antibody in mice and protects them from virus challenge. Am. J. Trop. Med. Hyg. 1991, 45, 159-167 25 Zhao, B., Prince, G., Horswood, R., Eckels, K., Summers, P., Chanock, R. and Lai, C.-J. Expression of dengue virus structural proteins and non-structural protein NSl by a recombinant vaccinia virus. J. Virol. 1987, 61, 4019-4022 26 Deubel, V., Kinney, R.M., Esposito, J.J., Cropp, C.B., Vorndam, A.V., Monath, T.P. and Trent, D. Dengue 2 virus envelope protein expressed by a recombinant vaccinia virus fails to protect monkeys against dengue. J. Gen. Virol. 1988, 69, 1921-1929 27 Haishi, S., Imai, H., Hirai, K., Igarashi, A. and Kato, S. Expression of envelope glycoprotein (E) of Japanese encephalitis virus by recombinant vaccinia virus. Acta Virol. 1989, 33, 497-503 28 Bray, M., Zhao, B., Markoff, L., Eckels, K.H., Chanock, R.M. and Lai, C.-J. Mice immunized with recombinant vaccinia virus expressing dengue 4 virus structural proteins with or without nonstructural

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29

30

31

32

33

34

35

36

37 33

39

40 41 42 43 44 45

46 47

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