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WO1992003161A1 - Proteines d'enveloppe de flavivirus avec pouvoir immunogene accru utilisables dans l'immunisation contre les infections virales - Google Patents

Proteines d'enveloppe de flavivirus avec pouvoir immunogene accru utilisables dans l'immunisation contre les infections virales Download PDF

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Publication number
WO1992003161A1
WO1992003161A1 PCT/US1991/006031 US9106031W WO9203161A1 WO 1992003161 A1 WO1992003161 A1 WO 1992003161A1 US 9106031 W US9106031 W US 9106031W WO 9203161 A1 WO9203161 A1 WO 9203161A1
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virus
protein
dengue
flavivirus
recombinant
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PCT/US1991/006031
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English (en)
Inventor
Ching-Juh Lai
Ruhe Men
Michael Bray
Lei-Ron Jan
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The United States Of America, Represented By The Secretary, United States Department Of Commerce
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Publication of WO1992003161A1 publication Critical patent/WO1992003161A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to flavivirus E proteins and their use in vaccines against flavivirus infection.
  • JEV Japanese encephalitis virus
  • Flaviviruses including the dengue virus and the JEV, contain only three structural proteins, that is, a capsid contain only three structural proteins, that is, a capsid protein (C) (mol. wt. 12-14 kd) which binds to the positive strand genomic RNA forming the nucleocapsid, and two membrane associated proteins termed the small membrane protein (M) (mol. wt. 7-8 kd) and the large membrane protein also called envelope glycoprotein (E) (mol. wt. 55-60 kd) (Stollar, V. 1969. Studies on the nature of dengue viruses. IV The structural proteins of type 2 dengue virus. Virology 39:426-438).
  • C capsid protein
  • M small membrane protein
  • E envelope glycoprotein
  • the envelope glycoprotein is the major virion antigen responsible for virus neutralization by specific antibodies and for several important antigenic properties such as binding to flavivirus-, dengue complex-, and type-specific antibodies (Clarke, D.H. 1960. Antigenic analysis of certain group B arthropod-borne viruses by antibody absorption. J. Exp. , Med. 111:21-32. Roehrig, J. T. , J. H. Mathew, and D. W. Trent. 1983. Identification of epitopes on the E glycoprotein of St. Louis encephalitis virus using monoclonal antibodies. Virology 128:118-126).
  • Dengue and other flavivirus E's also exhibit a hemagglutinating activity that is presumably associated with virus attachment to the cell surface and subsequent virus uncoding (Sweet, B. H. , and A. B. Sabin. 1954. Properties and antigenic relationships of hemagglutinins associated with dengue viruses. J. Immunol. 73;363-373).
  • the full-length dengue type 4 virus E sequence contains 494 amino acids including two hydrophobic regions at the C-terminus, 15 amino acids and 24 amino acids in length, separated by an arginine. These hydrophobic sequences may serve to interact with the lipid membrane during virus assembly.
  • Evidence from limited protease digestion of tick-borne encephalitis virus E glycoprotein suggest that the hydrophobic C-terminus is inserted into the lipid membrane exposing the bulk of the N-terminus of E on the virion surface.
  • the full-length E of dengue type 4 virus contains 12
  • N-terminal E contains both potential glycosylation sites.
  • E glycoproteins of 2 flaviviruses, West Nile virus and Kunjin virus lack glycosylation sites suggesting that N-glycosylation is not essential to the antigenic, structural and functional integrity of the flavivirus envelope glycoprotein (Coia, G. , M. D. Parker, G. Speight, M.E. Byrne, and E. G. Westaway. 1988.
  • results of epitope mapping with a library of monoclonal antibodies indicate that the antigenic structure of dengue E is similar to that of other flavivirus E , s that contain several distinct antigenic sites as defined by serological specificity, functional activity, and competitive binding assay (Henchal, E. A., J. M. McCown, D. S. Burke, M. C. Sequin, and W. E. Brandt. 1985. Epitopic analysis of antigenic determinants on the surface of dengue-2 virus using monoclonal antibodies. Am. J. Trop. Med. Hvg. 34:162-169; and Heinz, F. X. 1986. Epitope mapping of flavivirus glycoproteins. Advance in Virus Research Vol. 31, pp. 103-168, K.
  • the vaccinia virus recombinant system was also employed to separately express dengue E or NS1 glycoprotein (Bray, M. , B. Zhao, L. Markoff, K. H. Eckels, R. M. Chanock, and C. J. , Lai. 1989. Mice immunized with recombinant vaccinia virus expressing dengue 4 virus structural proteins with or without nonstructural protein NS1 are protected against fatal dengue virus encephalitis. J. Virol. 63:2853-2856; and Falgout, B., R. M. Chanock, and C. J. Lai. 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.
  • Immunization of mice with recombinant vaccinia virus expressing authentic dengue virus nonstructural protein NSl protects against lethal dengue encephalitis. J. Virol. 64: 4356-4363, 1990) .
  • mice with baculovirus recombinant expressed E and NSl also induced a similar level of resistance (Zhang, Y. M. , E. P. Hayes, T. C. McCarty, D. R. Dubois, P.L. Summers, K. H. Eckels, R. M. Chanock, and C. J. Lai. 1988. Immunization of mice with dengue structural proteins and nonstructural protein NSl expressed by baculovirus recombinant induces resistance to dengue encephalitis. J. Virol. 62:3027-3031).
  • v(C-M-E-NSl-NS2A) or v(93%E) or the baculovirus recombinant-infected cell lysate containing expressed E consistently failed to induce detectable antibodies to E or induced only a very low level of such antibodies in mice (Bray, M. , B. Zhao, L. Markoff, K. H. Eckels, R. M. Chanock, and C. J., Lai. 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. J. Virol. 63:2853-2856; and Zhang, Y. M. , E. P.
  • a vaccinia recombinant vaccine producing dengue virus antigens should share the characteristics of the vaccinia virus vaccine proven during successful global smallpox eradication campaign. That is, the vaccine should be safe, heat stable and easily administered, and have a low cost per dose.
  • the low immunogenicity of E constitutes a major obstacle to the development of an effective dengue vaccine produced by recombinant DNA technology.
  • the present invention relates to a vaccine for humans and animals against flavivirus infection comprising a recombinant virus expressing a
  • C-terminally truncated flavivirus envelope protein in an amount sufficient to induce immunity against the infection and a pharmaceutically acceptable carrier, wherein the truncated protein is intracellularly accumulated, or extracellularly secreted, or expressed on the outer membrane of infected cells.
  • the present invention relates to a vaccine for humans and animals against flavivirus infection comprising a recombinantly expressed truncated flavivirus envelope protein in an amount sufficient to induce immunity against the infection and a pharmaceutically acceptable carrier.
  • the present invention relates to a DNA construct comprising a DNA segment encoding a C-terminally truncated flavivirus protein and a vector, wherein the truncated protein is expressed intracellularly, extracellularly, or on the outer membrane of host cells expressing the construct.
  • the present invention relates to a recombinant virus encoding a C-terminally truncated flavivirus envelope protein which is expressed intracellularly, extracellularly, or on the outer membrane of host cells infected with the virus.
  • the present invention also relates to host cells productively infected with the recombinant virus of the present invention.
  • FIGURE 1 shows an analysis of dengue C-terminally truncated and the full-length envelope glycoproteins expressed by recombinant vaccinia viruses.
  • 35 S-methionine labeled lysates of CV-1 cells infected with various recombinant virus constructs were prepared and immunoprecipitated with a dengue virus hyperimmune mouse ascitic fluid (HMAF) . Each immunoprecipitate was divided into two aliquots: one was treated with endoglycosidase F (+) ; the other remained untreated (-) . Samples were subsequently analyzed on SDS- 12% polyacrylamide gel. The predicted length of the N-terminal envelope (E) sequence expressed by each recombinant is indicated.
  • HMAF dengue virus hyperimmune mouse ascitic fluid
  • HMAF precipitate of 35 S-methionine labeled lysate of dengue virus infected cells was used as dengue protein size markers (DEN) .
  • FIGURE 2 demonstrates the differential HMAF binding affinities of two truncated dengue E's.
  • the labeled lysates of cells infected with recombinants v(79%E) and v(81%E) were the same as described in FIGURE 1. Each lysate was divided into four equal aliquots: two were precipitated with HMAF in one- and three-fold concentrations. The other two precipitated with a rabbit anti-peptide serum directed against peptide 73 of the E protein (amino acid 259-272) also in one- and three-fold concentrations. E-specific protein bands are indicated. Other bands labeled present in the high molecular weight region in the precipitates with the rabbit serum, were apparently not related to dengue E.
  • FIGURE 3 shows the amino acid sequence involved in transition of dengue E antigenic structure.
  • 35 S-methionine labeled lysates were prepared from CV-1 cells infected with recombinant vaccinia viruses that expressed 79%E, 79%E-R, 79%E-RK, 79%E-RKG, 79%E-RKGS, and 79%E-RKGSS. Equal aliquots of each lysate were precipitated separately with HMAF and with rabbit anti-peptide 73 serum followed by analysis on SDS-polyacrylamide gel. M shows sizes of protein markers in kilodaltons.
  • FIGURE 4 shows an analysis of dengue E's in intracellular and extracellular fractions.
  • CV-l cells were infected with various recombinants expressing C-terminally truncated dengue E's of the sizes indicated, or were not infected (Mock) . At 18 hours after infection, cells were labeled with 35 S-methionine for 6 hours.
  • the intracellular fraction (INT) and the extracellular (medium) fraction (EXT) were prepared and immunoprecipi- tated with HMAF for separation on SDS-polyacrylamide gel. Dengue E's in the gel lanes are indicated.
  • the recombinant construct 100%E contained dengue DNA coding for the full-length dengue E plus 4 amino acids of NSl.
  • FIGURES 5A-5D show detection of truncated E's of dengue virus on the surface of recombinant vaccinia virus infected cells.
  • An immunofluorescence assay on live cells was performed.
  • Figure 5A shows cells infected with v(79%E-RKG) ;
  • Figure 5B shows cells infected with v(81%E) ;
  • Figure 5C shows cells infected with v(59%E) ;
  • Figure 5D shows cells infected with v(100%E) .
  • FIGURE 6 shows an analysis of intracellular and secreted dengue E's by endoglycosidase digestion.
  • Immunoprecipita_3s were digested with endoglycosidase H (H) , or endoglycosidase F (F) , or mock-digested (-) .
  • the intracellular fraction (INT) or the extracellular fraction (EXT) of the recombinant E products was prepared for this analysis.
  • M shows the molecular sizes of marker proteins in kilodaltons.
  • D is the immunoprecipitate of dengue virus proteins for size comparison.
  • FIGURES 7A and 7B display the relationship of protec ⁇ tive efficacy of vaccinia virus dengue E recombinants to overall antigenicity of expressed E products.
  • the top panel shows relative binding of the expressed E products to dengue virus hyperimmune mouse ascitic fluid (HMAF) as detected by radio-immunoprecipitation. On a scale of 1 to 4, the highest number was assigned to the E products which exhibit high HMAF binding affinity, and the lower numbers (1 and 3) to E's which bind less efficiently to HMAF.
  • the lower panel shows the cumulative protection rates as expressed by percent survival following dengue virus challenge of mice immunized with various vaccinia recombinants expressing 9-100% dengue E's. The results were derived from 4 separate mouse protection studies. The total number of mice tested for each recombinant is indicated.
  • FIGURE 8 shows an analysis of E antibodies in sera of immunized mice by radio-immuno precipitation.
  • Lanes 1, 2, 3, 4, 5, and 6 are serum samples from individual mice immunized once with v(79%E-RKG) .
  • Lanes 4', 5' and 6' indicates serum samples collected from the same animals 5 days later.
  • Lanes in the pooled sera are: A, mice infected with vSC8 (Control vaccinia virus) ; B, with v(59%E); C, with v(79%E-RKG) ; D, with v(81%E) ; and E, with v(100*%E) .
  • HMAF at 1/40, 1/80, or 1/160 dilution was used for precipitation of the same labeled dengue antigen preparation.
  • FIGURE 9 shows the alignment of amino acid sequences in the antigenically critical region dengue type 4 and three other dengue serotype E glycoproteins.
  • the 26 amino acid sequence (positions 373-398) of denge type 4 glycoprotein was compared with the corresponding E sequences of dengue type 1, type 2, and type 3 viruses.
  • Arg (R) at position 392 at the C-terminus of dengue type 4 80% E has been shown to be critical for the antigenic structure displaying a high affinity binding by HMAF (in this invention) .
  • FIGURE 10 shows the analysis of dengue type 4 virus 80% E expressed by recombinant baculovirus.
  • Insect SF9 cells infected with recombinant baculovirus were radio- labeled for 2 hours and the labeling medium was replaced with serum-free Grace medium. Aliquots of the medium fluid were collected at various times for immunoprecipitation of extracellular 80% E. An equivalent aliquot of the cell update was also immunoprecipitated to determine the amount of 80% E that remained intracellularly. The six-hour medium sample was also analyzed without immunoprecipitation. The molecular weight markers (lane M) are shown on the right.
  • FIGURE 11 shows an alignment of sequences in the antigenically critical region of dengue type 4 and Japanese encephalitis virus envelope glycoproteins.
  • the 26 amino acid sequence (positions 373-398) of dengue type 4 E glycoprotein was compared with the corresponding sequence (positions 379-404) of JE E glycoprotein.
  • Arg at position 392 of dengue type 4 E has been shown to be critical for the antigenic structure (see FIGURE 3) .
  • a conserved amino acid found between the two viruses is indicated by a dot in the JE sequence.
  • FIGURE 12 shows an analysis of E glycoproteins expressed by recombinant vaccinia virus.
  • CV-l cells infected with recombinant v (JE, 100% E) or v (JE, 80% E) were labeled with 35 S-methionine.
  • the cell lysate was immunoprecipitated using JE HMAF.
  • Both recombinant expressed E's were further digested with endo H (lane H) or endo F (lane F) to analyze the extent of glycosylation.
  • the medium fraction of v (JE, 80%) ' or v (JE, 100%) infected cells was similarly analyzed. Marker proteins shown in lane JE are JE virus proteins.
  • FIGURE 13 shows the JE virus C-terminally truncated E expressed on the cell surface and extracellularly secreted.
  • A Infection of CV-1 cells and radio-labeling were the same as in FIGURE 12. JE E produced in the lysate, in the medium fluid, or on the cell surface was analyzed on a polyacrylamide gel. Note that the long exposure of the film was intended to reveal in the surface lane a labeled protein band identified as 100% E based on co-migration with the full-length E indicated by the long arrow. C-terminally truncated E (80% E) indicated by the short arrow is present in the medium and on the surface. Other minor bands on the gel are background since they are also present in the corresponding fractions of the vSC8 control.
  • B Following a 2 hr radio-labeling, medium from v (JE, 80% E)-infected cells was collected at various times indicated and analyzed as described herein.
  • FIGURES 14A and 14B show an analysis of sero-response by radio-immunoprecipitation.
  • Serum samples from CD-I mice used for the challenge study and from the three inbred strains of mice used for the immunogenicity study were analyzed by radio-immunoprecipitation.
  • the radio- labeled immunoprecipitates were separated on polyacryl- amide gels.
  • Sera of mice immunized with v (JE, 100% E) which expresses the full-length E are shown on the left; sera of mice immunized with v (JE, 80% E) which expresses the C-terminally truncated E are shown on the right.
  • Dengue E glycoprotein like other flavivirus E glycoproteins, plays an important role in various stages of viral infection and the development of protective immunity.
  • the sequence of the dengue type 4 virus envelope protein (E) was analyzed by systematic C-terminal deletion and expression of the resulting truncated E product in a vaccinia virus recombinant in an attempt to delineate E sequences responsible for inducing resistance in mice and to improve the immunogenicity of this major viral protein antigen.
  • E dengue type 4 virus envelope protein
  • the present invention relates to DNA constructs encoding a truncated flavivirus E protein, for example, dengue type 4, dengue type 2 or Japanese encephalitis virus E.
  • DNA constructs to which the present invention relates comprise a vector and a DNA segment encoding a sufficient amount of the N-terminus sequence of a flavivirus E protein so that expression of the protein alters its intrac lular processing pathway resulting in accumulation on the outer membrane of the cell.
  • the truncated protein may also be secreted extracellularly.
  • the DNA segment may encode at least 80% of the N-terminus sequence of dengue type 4 virus an E protein, preferably between 80 and 81%.
  • 80% E refers to 79% of the N-terminus sequence of dengue type 4 virus E plus R, RK, RKG, RKGS, or RKGSS (amino acids designated by the standard single letter code) .
  • flavivirus E's refer to the E molecules corresponding in size to the dengue type 4 8C E sequence.
  • Suitable vectors for use in the present invention include, but are not limited to, the vaccinia virus vector and baculovirus vector.
  • the DNA constructs of the present invention are used to generate recombinant viruses such as, for example, recombinant vaccinia viruses and recombinant baculoviruses using methods known in the art.
  • the present invention further relates to recombinant viruses encoding the truncated E protein of the present invention.
  • a recombinant vaccinia virus encoding a truncated dengue type 4, or type 2, or JEV E protein preferably. encoding about 80% E
  • JEV E protein preferably. encoding about 80% E
  • the present invention relates to eukaryotic host cells producing the truncated E protein.
  • Suitable host cells include, but are not limited to, mammalian cells such as, CV-1 and TK " 143 cells, and insect cells such as, Spodoptera frugiperda cells.
  • the recombinant virus is a baculovirus
  • the host cell must be an insect cell.
  • Host cells are infected with the recombinant viruses of the present invention and are cultured under conditions allowing expression of the encoded truncated E protein. Some of the expressed truncated flavivirus E protein is retained on the outer membrane of the cell and some of it is also secreted from the cell.
  • mammalian cells infected with a recombinant vaccinia virus expressing the truncated dengue type 4 E or JEV E protein were shown to accumulate the truncated protein on their cell surface by indirect immunofluorescence and to secrete the truncated protein extracellularly by radio immunoprecipitation.
  • insect cells infected with a recombinant baculovirus of the present invention also express the truncated protein into the medium fluid.
  • the present invention also relates to recombinantly produced truncated flavivirus E protein and to antibodies specific therefor.
  • the recombinantly produced E protein is between 80 and 81% of the N terminus sequence of the naturally occurring E protein, or an allelic variation thereof.
  • the recombinantly produced protein may be glycosylated or unglycosylated.
  • One skilled in the art without undue experimentation, can easily modify, partially remove or completely remove the natural glycosyl groups from the E protein of the present invention using standard methodologies.
  • the flavivirus E proteins of the present invention are the flavivirus E proteins of the present invention.
  • mice inoculated with recombinant vaccinia virus encoding truncated dengue virus E or truncated JEV E were protected against subsequent viral challenges.
  • the increased immunogenicity in the mice correlated with the increase in antibody production.
  • the passive transfer of sera from immunized mice conferred protection against viral challenge to the recipient mice.
  • dengue type 4 50% E which was extracellulary secreted did not show an increase of immunogencity and protective efficacy. Accordingly, surface expression of the truncated form of the E protein appear responsible for the enhanced immunity.
  • the present invention further relates to vaccines for use in humans and animals such as pigs and horses against flavivirus infection.
  • Protective antibodies against flaviviruses can be raised by administering a vaccine containing the recombinant virus of the present invention or the purified truncated E protein of the present invention.
  • Vaccines of the present invention can also include effective amounts of immunological adjuvants known to enhance an immune response.
  • One vaccine of the present invention contains the recombinant vaccinia virus of the present invention, or its derivative using a strain of vaccinia virus certified for humans, in an amount sufficient to induce production of the encoded flavivirus E protein causing an immune response against flavivirus infection.
  • the vaccine can be administered by an intradermal route.
  • Another vaccine of the present invention contains the truncated protein produced by host cells of the present invention, for example, Sf9 insect cells.
  • the truncated E protein n be delivered in a pharmacologically acceptable vehicle to induce an antibody response.
  • the truncated protein preferably comprising between 80 and 81% of the N-terminus sequence, is expressed by the recombinant virus of the present invention (preferably, a recombinant baculovirus) during an infection by the recombinant virus and can be isolated from the cell lysate or the medium fluid using standard methods in the art.
  • the present inventors have shown that appropriately truncated E constructs of flaviviruses are candidate vaccines for the prevention of disease. Increased immunity has been exemplified with the use of truncated E of the dengue type 2, dengue type 4 and JEV flaviviruses.
  • Dengue Virus Experiments (1) Construction of dengue virus DNA's coding for full- length and C-terminally truncated E's.
  • the dengue virus cDNA fragment that codes for the putative 15 amino acid N-terminal signal and the entire E sequence except the last 39 amino acids at the C-terminus was obtained earlier (Bray, M. , B. Zhao, L. Markoff, K. H. Eckels, R. M. Chanock, and C. J. Lai. 1989. Mice immu ⁇ nized with recombinant vaccinia virus expressing dengue 4 virus structural proteins with or without nonstructural protein NSl are protected against fatal dengue virus encephalitis. J. Virol. 63:2853-2856).
  • An extended DNA fragment coding for the N-terminal signal the entire E plus the first 30 amino acids of the downstream NSl nonstructural protein was constructed from the previously derived DNA fragment terminating at the Sst I site at nucleotide 1931 of the dengue type 4 viral sequence and the Sst I - Sau 3A DNA fragment (nucleotides 1931-2592) using the shared Sst I site for joining.
  • This extended E DNA construct was inserted into the Bgl II site of inter ⁇ mediate vaccinia vector plasmid pSCll (Chakrabarti, S., K. Brechling, and B. Moss. 1985.
  • Vaccinia virus expression vector coexpression of ⁇ -galactosidase provides visual screening of recombinant virus plaques. Mol. Cell. Biol. 5:3403-3409; and Falgout, B., R. M. Chanock, and C. J. Lai. 1989. Proper processing of dengue virus nonstruc ⁇ tural glycoprotein NSl requires the N-termin hydrophobic signal sequence and the downstream nonstructural protein NS2A. J. Virol. 63:1852-1860). At the downstream Bgl II site regenerated in the recombinant DNA construct a linker sequence was inserted consisting of oligo 2196 (GATCCTAGCTAGCTAGGTACC) (SEQ. ID.
  • oligo 2197 (GATCGGTACCTAGCTAGCTAG) (SEQ. ID. NO:2) that contained stop codons in all three reading frames followed by a Kpn I cleavage site.
  • the insertion of this linker sequence destroyed the joining Bgl II site leaving the unique upstream Bgl II site in the recombinant plasmid.
  • the extended DNA sequences cleaved from the plasmid by Bgl II and Kpnl were digested with Bal 31 and the progressively shortened DNA fragments were ligated to a second Kpn 1 linker sequence consisting of oligo 2246 (TGAATGAATGAGATCTGGTAC) (SEQ. ID. NO:3) and oligo 2247 (CAGATCTCATTCATTCA) (SEQ. ID. NO:4) that contained stop codons in all three reading frames.
  • DNA coding for a truncated E terminating at a specific amino acid residue such as 79%E plus -R, -RK, -RKG, -RKGS, or -RKGSS was constructed using the polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • Oligo 2552 (AGATCTGGTACCTAGGAACTCCCTTTCCTGAA) (SEQ. ID. NO:6); 79%E-RKGS, oligo 2553
  • CV-1 and Human TK ⁇ 143 cells were grown and propagated in minimum essential medium plus 10% fetal calf serum (MEM10) .
  • Wild type vaccinia virus strain WR was used and recombinant vaccinia viruses were constructed according to the procedure described earlier (Chakrabarti, S., K. Brechling, and B. Moss. 1985.
  • Vaccinia virus expression vector coexpression of j8-galactosidase provides visual screening of recombinant virus plaques. Mol. Cell. Biol. 5:3403-3409; and Zhao, B., E. Mackow, A. Buckler-White, L. Markoff, R. M. Chanock, C. J. Lai, and Y. Makino. 1986. Cloning full-length dengue type 4 virus DNA sequences: analysis of genes coding for structural proteins. Virology 155:77-88). Recombinant vaccinia virus vSC8 that contained a lacZ gene insert was used as a control.
  • Fluid medium of infected cell cultures was collected after the labeling period and analyzed directly by immunoprecipitation.
  • Infected cells were disrupted in a volume of RIPA buffer equivalent to the volume of the fluid medium.
  • Dengue hyperimmune mouse ascitic fluid (HMAF) prepared against dengue 4 virus strain 814669 was used at a dilution of 1:5 or 1:20 for immunoprecipitation of the labeled lysates.
  • a rabbit anti-serum raised against a dengue type 4 virus E peptide (peptide 73, amino acids 259-272 of the E sequence) was also used at a dilution of 1:5 or 1:20 for immunoprecipitation.
  • E-specific antibodies in individual or pooled sera of immunized mice were analyzed by immunoprecipitation of a 35 S-labeled lysate of dengue virus infected LLCMK 2 cells as described previously (Zhang, Y. M. , E. P. Hayes, T. C. McCarty, D. R. Dubois, P. L. Summers, K. H. Eckels, R. M. Chanock, and C. J. Lai. 1988. Immunization of mice with dengue structural proteins and nonstructural protein NSl expressed by baculovirus recombinant induces resistance to dengue encephalitis. J. Virol. 62:3027-3031). (6) . Indirect immunofluorescence assay.
  • Confluent CV-1 cells in a chamber slide were infected with recombinant virus at a low multiplicity (less than 10 pfu/well) .
  • MEM minimum essential medium
  • acetone fixed cells
  • HMAF fluorescein conjugated rabbit anti-mouse immunoglobulins
  • dengue type 2 80%E was shown to be more immunogenic than the full length E
  • recombinant baculovirus expressing dengue type 2 80%E was similarly constructed. These recombinant baculoviruses were designated b(DEN4, 80%E) and b(DEN2, 80%E) , respectively.
  • FIGURE 9 shows the alignment of the 26 amino acid- sequence (positions 373-398) of dengue type 4 E glycoprotein with the corresponding sequences of three other dengue serotype E glycoproteins. Arg at position 392 of dengue type E has been shown to be critical for the antigenic structure. A conserved amino acid found dengue type 4 and a given dengue serotype is indicated by a dot. (8) . Analysis of extracellularly secreted dengue virus 80%E expressed by recombinant baculovirus.
  • insect sf9 cells were infected with recombinant baculovirus b(DEN4, 80%E) or b(DEN2, 80%E) . Two days after infection, the sf9 cells were radio-labeled for 2 hr and the labeling medium replaced with serum-free Grace medium (GIBCO Laboratories, Grand Island, N.Y.). Culture fluid was sampled at various times to determine the amount of the E product secreted relative to that detected in the cell lysate.
  • dengue virus type 4 80%E detected in the medium increased with time during the 6-hr chase period. At six hours following the labeling, approximately 40% of the labeled E product was found in the medium. The secreted E product appeared to represent the major protein component in the medium as shown in the gel lane (far right) in which the fluid sample was not immunoprecipitated. This finding indicates that a significant fraction of baculovirus-expressed dengue type 4 virus 80%E is secreted extracellularly.
  • JEV Japanese Encephalitis Virus
  • JEV strain SA-14 was used as the challenge virus in the studies.
  • a seed stock of JEV SA-14 challenge virus was prepared from the brain homogenate after two passages in suckling mouse brains.
  • JEV JAOArS982 was used as the source for cDNA needed to construct the recombinant virus.
  • Polymerase chain reaction (PCR) was performed to derive appropriate JE virus cDNA fragments for expression by recombinant vaccinia virus.
  • template DNA was clone S-22 that was used to obtain the coding sequence for PreM, E and portions of flanking C and NSl (Sumiyoshi, H. , C. Mori, I. Fuke, K. Morita, S. Kuhara, J. Kundou, Y. Kikuchi, H. Nagamatsu, and A. Igarashi. 1987. Complete necleotide sequence of the Japanese encephalitis virus genome RNA. Virology 161:497-510)).
  • the positive strand primer was oligonucleotide (oligo) AAC AAC GGA TCC ATG GTG GTG TTT ACC ATC CTC CTG (SEQ. ID.
  • the primer introduces the flanking Bam HI cleavage sequence, an initiation codon preceding the coding sequence for the 15 amino acid hydrophobic signal preceding the N-terminus of E.
  • the negative strand primer was Oligo CAC ATG GAT CCT AAG CAT GCA CAT TGG TCG CTA A (SEQ. ID. NO:13) which provides the Bam HI sequence and a translation stop following the last coding sequence of E.
  • the JEV DNA fragment coding from the 80%E was constructed using the same positive strand primer as shown above and oligo GCC TAG GGA TCC TCA AGC TTT GTG CCA ATG GTG GTT (SEQ. ID. NO:14) was the negative strand primer.
  • This negative strand primer contains a stop codon and the Bam HI cleavage sequence following the coding sequence for alanine at position 399.
  • Both predicted PCR products were isolated after separation on an agarose gel, cleaved by Bam HI digestion, and inserted into the intermediate transfer vector pSCll (Bgl II) . The structures of both recombinant DNA constructs were verified by sequencing across the Bam HI-Bgl II junctions.
  • radio- labeled cells in a ⁇ ⁇ -well plate were placed in 0.5 ml MEM containing HMAF at 1:100 dilution for 1 hr at 37° C. After extensive washing with MEM to remove excess HMAF that was not bound to E on the cell surface or remained in the medium, cells were lysed in RIPA buffer (1% sodium deoxycholate, 1% Nonidet P40, 0.1% sodium dodecyl sulfate, 0.1M Tris hydrochloride, pH 7.5, 0.15 M NaCl) and the lysate used directly for precipitation with Pansorbin beads (Calbiochem-Boehringer) .
  • RIPA buffer 1% sodium deoxycholate, 1% Nonidet P40, 0.1% sodium dodecyl sulfate, 0.1M Tris hydrochloride, pH 7.5, 0.15 M NaCl
  • the medium fraction collected following the labeling period was made into RIPA buffer and analyzed by precipitation with JEV HMAF.
  • Endogylcosidase F (Endo F)
  • mice outbred strain CD-I were inoculated at an intraperitoneal site with 3 x 10 6 pfu of recombinant vaccinia virus expressing JEV E described above. The mice were again inoculated 14 days later. One week after the second inoculation, mice were bled and the following day challenged intraperitoneally with 100 LD 50 of JE virus SA-14 in 0.3 ml. As part of the challenge, mice were also intracerebrally inoculated with 0.03 ml virus-free MEM. JEV infected mice were observed for symptoms of viral encephalitis and death daily for 3 weeks. Control mice were immunized with recombinant vaccinia virus vSC8.
  • mice In the immunogenicity study, three inbred strains of mice (BALB/c, C57BL/6, and C3H/He) at 3 weeks of age were immunized intraperitoneally twice with 3 x 10 6 pfu/dose of recombinant vaccinia virus at 14 day intervals. Blood samples collected one week after the second inoculation. (12) . Analysis of sera using radioimmumnoprecipitation and ELISA.
  • JE SA-14 virus-infected cells labeled with 35 S-methionine were lysed and the lysate was used as the labeled antigen.
  • 3 ⁇ l of individual or pooled mouse sera was added to 20 ⁇ l of the labeled lysate and the mixture incubated on ice for 2 hr.
  • the immunoprecipitate was analyzed on polyacrylamide gels as described above.
  • the antigen in the solid phase was sucrose gradient-purified JEV SA-14 virus, and the second antibody was peroxidase-conjugated goat anti-mouse IgG (Kirkegaard and Perry Laboraties, Gaithersburg, MD) .
  • Sera at 1:1000 dilution was the starting concentration and serially two-fold diluted for testing.
  • recombinant DNA's coding for the full-length E (100%) , or 99%, 94%, 88%, 81%, 79%, 70%, 66%, 59%, 50%, 37%, 27%, 19%, or 9% of the N-terminal sequence of E were constructed.
  • Each recombinant plasmid was sequenced to determine the truncation site in order to identify the sequence coding for the C-terminal amino acids of the E product.
  • the predicted C-terminal amino acid sequences are shown below in Table I.
  • Recombinant vaccinia viruses containing this series of E DNA's were then constructed and used for infection of CV-1 cells.
  • 35 S-methionine labeled lysates were prepared from infected cells, precipitated with HMAF, and the precipitates analyzed by polyacrylamide gel electrophoresis.
  • Each of the eight recombinants that contained 59% or more of the coding sequence produced a dengue E specific protein of the predicted size (see FIGURE 1) .
  • Digestion with endoglycosidase F reduced the molecular size of each product to the value estimated for the unglycosylated protein.
  • peptide 73 anti-serum precipitated both E's to the same extent suggesting that 7J%E, was as stable as 81%E, but was not precipitated efficiently by HMAF (see FIGURE 2) .
  • an increased level of detection by peptide 73 antiserum was observed for 70%, 66%, or 59%E.
  • Peptide 73 antiserum did not precipitate 50%E or other smaller E species lacking the peptide sequence.
  • HMAF efficiently precipitated 79%E-R, and also the other longer E species, but not 79%E.
  • peptide 73 antiserum detected 79%E, 79%E-R, and other larger E products with similar efficiency (see FIGURE 3) .
  • the arginine residue immed. ely downstream of the C-terminus of 79%E is required ⁇ or the formation and/or maintenance of the native conformation of E and thus conformational E epitopes recognized by HMAF.
  • the arginine residue at position 392 plays a critical role in the proper folding of dengue E during or after polypeptide synthesis presumably through a charged amino acid interaction between this arginine and the other regions of the molecule.
  • This position in the linear E sequence appears to represent a transition between the two classes of E's that are distinguishable by their binding affinities to HMAF (FIGURE 3) . Since this arginine is located C-terminal of the last cysteine residue, it is likely that this charged amino acid influences the folding of the E molecule during nascent synthesis so that cysteine residues can be brought into close proximity for disulfide bond formation which is a prerequisite for maintenance of a stable configuration. Of interest, this arginine corresponds in position to the trypsin cleavage site of tick-born encephalitis virus E glycoprotein at position 395 suggesting that this residue in the native E structure is accessible to the enzyme (Mandl, C. W. , F.
  • CV-1 cells were infected with a recombinant expressing 59%E, 70%E, 79%E-R, 79%E-RKG, 81%E, 88%E, or 100%E. After a 6 hr period of labeling with 35 S-methionine the cell lysate and the medium fractions were separately analyzed by immunoprecipitation with HMAF (see FIGURE 4) .
  • HMAF detected the synthesis of each of the E's in the cell lysate fraction, although 79%E-R or 79%E-RKG and the three other larger E species were more efficiently precipitated than 79%E, or other smaller E's as observed earlier. 59%E, 66%E, 70%E, 79%E-R, and 79%E-RKG were detected in significant amount in the fluid medium ranging from 10-50% of the total labeled product. Similarly, 30-50% of label in 79%E-RK, 79%E-RKGS, or 79%E-RKGSS was also detected in the medium fraction during the 6 hr labeling period.
  • This hydrophobic structure resembles the C-terminal anchor of many surface glycoproteins in that a hydrophobic trans ⁇ membrane domain of length sufficient to span the lipid bilayer (usually 20-30 amino acids) is followed by a charged cytoplasmic domain of varying length. It appears that this internal sequence of full-length
  • the immunoglobulin ⁇ chains of membrane-bound and secreted IgM molecules differ in their C-terminal segments.
  • the sequence of 3 amino acids in the C-terminal domain of 79%E-RKG, or especially, the sequence of one charged amino acid (Arg) in the C-terminal domain of 79%-R is considerably shorter than the cytoplasmic domains of other viral integral membrane proteins and may affect its stable association with membranes as a similar finding has been described (Kilpatrick, D. R. , R. V. Srinivas, E. B. Stephens, and R. W. Compans. 1987. Effects of deletion of the cytoplasmic domain upon surface expression and membrane stability of a viral envelope glycoprotein. J. Biol. Chem. 262:16116- 16121) . Perhaps for this reason, 79%E-RKG or 79%E-R failed to become firmly anchored on the membrane and, as a consequence, a fraction of the expressed protein was secreted.
  • FIGURE 5 shows the result of this assay for several representative recombinants including v(59%E) and v(79%E-RKG) , that expressed extracellular E's, and v(81%E) and v(100*%E) , whose E product was not secreted extracellularly.
  • Live cell immunofluorescence tests indicated that the E product of v(79%E-RKG) was present in high concentration at the cell surface, whereas v(81%E) infected cells exhibited considerably less cell surface antigen.
  • arginine at position 392 of the E sequence alters this molecule so that it becomes stably associated with the cell membrane and accumulates in high concentration at this site.
  • arginine at position 392 is also critical for the formation and maintenance of the native antigenic structure of E and for efficient secretion from recombinant virus infected cells.
  • 59%E and 79%E-RKG were secreted extracellularly only 79%E-RKG was detected on the cell surface.
  • 81%E accumulated in low concentration on the cell surface but was not secreted. Finally, 100%E was detected only inside recombinant virus infected cells.
  • the intracellular fraction of 59%E was sensitive to digestion by endo H or endo F indicating that its carbohydrate moiety was of the mannose-rich type that is added during the first stage of glycosylation during protein synthesis (see FIGURE 6) .
  • extracellular 59%E was completely resistant to endo H digestion. This indicates that 59%E had entered the secretory pathway through the Golgi apparatus where endo H-resistant carbohydrates were added prior to secretion into the medium.
  • FIGURE 6 also shows that the extracellular 79%E-RKG, similar to extracellular 59%E, was completely resistant to endo H, whereas the intracellular form of 79%E-"iKG was partially sensitive to endo H. In contrast, intracellular full-length E was completely sensitive to endo H digestion.
  • N-terminal sequence was evaluated by challenging immunized mice with dengue type 4 virus intracerebrally.
  • a summary of 4 separate immunization-challenge studies is presented in FIGURE 7.
  • the protection rate, shown as percent survival, represents the number of survivors relative to the number of immunized mice.
  • mice immunized with v(79%E-RKG) or with a recombinant expressing 81%, 88%, 94%, 99%, or 100%E were completely protected or almost completely protected against dengue virus challenge, ie., overall protection rates of 94-100%.
  • mice immunized with v(79%E) , or a recombinant expressing a smaller product ie. , 70%, 66%, 59%, 50%, 37%, 27%, or 19%E
  • mice immunized with v(59%E) or with a recombinant expressing 81%, 88%, 94%, 99%, or 100%E were completely protected or almost completely protected against dengue virus challenge, ie., overall protection rates of 94-100%.
  • mice immunized with v(79%E) , or a recombinant expressing a smaller product ie. , 70%, 66%, 59%, 50%, 37%, 27%, or 19%E
  • mice immunized with v(79%E-RKG) failed to develop detectable E antibodies in response to immunization.
  • each of the mice immunized with v(79%E-RKG) developed a moderate to high level of E antibodies.
  • not all groups of mice that exhibited complete or almost complete resistance to virus challenge developed such an E antibody response to immunization.
  • mice immunized with 81%E developed a low level of E antibodies while mice immunized with 100%E failed to develop detectable E antibodies (see FIGURE 8) .
  • mice Protection of mice against dengue virus challenge following passive transfer of sera from donor mice immunized with recombinant vaccinia virus expressing the full-length and C-terminally truncated E's Serum donor mice Response to dengue Protection immunized with challenge no. of rate (%) mice that survived relative to no. of mice that received sera
  • VSC8 * 1/10 10 Among the truncated E molecules that were able to bind antibodies in dengue virus HMAF efficiently, only 79%E-RKG was highly immunogenic when tested for induction of E antibodies. This truncated E was unique in being expressed in high concentration on the surface of infected cells. 79%E-RKG was also secreted extracellularly but other truncated E constructs that were also secreted efficiently did not induce a detectable E antibody response. This suggests that cell surface expression of 79%E-RKG was responsible for its enhanced immunogenicity. However, it is not possible to rule out the importance of a unique form of secreted E. The properties of 81%E were also consistent with the importance of cell surface expression of E in immunogenicity.
  • E constructs of other flaviviruses may also be suitable for the prevention of disease.
  • E sequence among flaviviruses amino acid homology among dengue viruses of different serotype ranges from 62 to 70% while homology among different flaviviruses ranges from 40-50%.
  • cysteine residues suggests conservation of the 3-dimensional structure of E.
  • the present inventors have recently evaluated the applicability of this strategy for another dengue virus serotype by constructing dengue type 2 virus (PR159, SI strain) truncated E similar in size to 79%E-RKG of dengue type 4 virus. Immunization with the recombinant vaccinia virus expressing this C-terminally truncated E of dengue type 2 virus induced solid resistance in mice to challenge with type 2 virus. In contrast, immunization with a recombinant expressing the full-length E of dengue 2 virus induced only partial protection at a survival rate of less than 50% whereas unprotected animals all died following dengue challenge.
  • JEV cDNA fragment that coded for the N-terminal 80% E terminating at Ala at position 399 was constructed.
  • a cDNA fragment that codes for the full-length JEV E was also prepared. These DNA fragments were used to construct a recombinant vaccinia virus expressing the N-terminal 80% sequence of E designated v (JE, 80%E) or a recombinant vaccinia virus expressing the full-length E, designated v(JE, 100%E) as described above.
  • v(JE, 100%E) produced a protein of 53 kilodaltons (kd) similar in size to the E glycoprotein of
  • endo H reduced the molecular size of the E product by 2-3 kd, a value consistent with the predicted structure that the full-length E protein product contained a mannose-rich carbohydrate side chain.
  • the full-length E glycoprotein was not detected in the medium fraction of recombinant virus infected cells.
  • HMAF precipitated a 42 kd protein which was the size predicted for 80%E.
  • 80%E contained a carbohydrate moiety of the mannose-rich type as it was sensitive to endo F or endo H digestion.
  • FIGURE 13 show the analysis of the full-length and truncated JEV E's in the immunoprecipitates by polyacrylamide gel electrophoresis.
  • Full-length JEV E was predominantly found in the cell lysate fraction, only a low level (1-5%) was detected on the cell surface.
  • Other labeled bands present in the immunoprecipitates were presumably background since they were also seem in the vSC8 control.
  • JEV 80%E was readily detected on the cell surface in an amount varying from 10-25% of 80%E in the cell lysate dependent on experiments. In this study, the amount of 80%E secreted into the medium was estimated to be 6-10% of the total labeled 80%E product. (13) .
  • the levels of E antibodies were examined by the intensities of labeled E in the precipitates after separation on polyacrylamide gels (FIGURE 14) .
  • the levels of E antibodies developed following immunization with v(JE, 100%E) varied among individual mice and E specific antibodies were low or not detected in 8 of the 14 serum samples.
  • mice immunized with v(JE, 80% E) contained E antibodies that were detected at a uniformly high level with the exception of two sera in which E antibodies were low or not detected.
  • the high level of E antibodies developed following immunization with v(JE, 80% E) appeared to correlate with the increased protection rate.
  • Such variation of antibody response among individual CD-I mice was consistently observed in both protection experiments. Since all CD-I mice were inoculated twice with 3 x 10 6 pfu/dose of a recombinant virus, the variability of antibody response might stem from the background genetic heterogeneity of outbred mice.
  • inbred mice strain C57BL/6 of the H2-b haplotype strain BALB/c of the H2-d haplotype, and strain C3H/He of the H2-k haplotype were used for immunization with v(JE, 100% E) , or v(JE, 80% E) .
  • Sera from immunized inbred mice were similarly analyzed by radio-immunoprecipitation as described. It can be seen in FIGURE 14 that E antibodies were detected at a uniformly high level in C57BL and BALB/c mice immunized with v(JE, 80% E) .
  • mice immunized with v(JE, 100% E) developed a low level of E antibodies.
  • 80% E induced a more uniform and a higher level of antibody response than did the full-length E in several strains of inbred mice tested.
  • Antibody response to 80% E JE was highest in BALB/c mice of the H-2d Haplotype and lowest in C3H/He mice belonging to the H2-K Haplotype.
  • the inventors also performed ELISA and a plaque- reduction neutralization test to analyze the level of E antibodies in sera of mice in order to further characterize the antibody response to immunization.
  • Pooled sera from CD-I mice that were immunized with v(JE, 100%E) , or v(JE, 80%E) and pooled sera from each strain of inbred mice similarly immunized were tested.
  • Table IV antibodies in sera of CD-I mice immunized with v(JE, 80% E) showed an 8-fold increase of the ELISA titer compared to antibodies in pooled sera of mice immunized with v(JE, 100% E) .
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:l: GATCCTAGCT AGCTAGGTAC C
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • Xi SEQUENCE DESCRIPTION: SEQ ID NO:3: TGAATGAATG AGATCTGGTA C
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:5: CGTTTGCCAT ACGCTCACAG
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • Xi SEQUENCE DESCRIPTION: SEQ ID NO:9: AGATCTGGTA CCTATTTCCT GAACCAATGG AG
  • MOLECULE TYPE DNA (genomic)
  • Xi SEQUENCE DESCRIPTION: SEQ ID NO:10: AGATCTGGTA CCTACCTGAA CCAATGGAGT GT
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • Xi SEQUENCE DESCRIPTION: SEQ ID NO:12: AACAACGGAT CCATGGTGGT GTTTACCATC CTCCTG
  • MOLECULE TYPE DNA (genomic)
  • Xi SEQUENCE DESCRIPTION: SEQ ID NO:13: CACATGGATC CTAAGCATGC ACATTGGTCG CTAA
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO:14: GCCTAGGGAT CCTCAAGCTT TGTGCCAATG GTGGTT

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Abstract

L'invention se rapporte à des protéines d'enveloppe de flavivirus tronquées au niveau de leur terminaison C, qui constituent en grosseur 80 à 81 % de la protéine initiale et qui ont un pouvoir immunogène plus grand que les mêmes protéines entières non tronquées; à des virus recombinants qui codent pour une telle protéine tronquée, ainsi qu'à des cellules hôtes infectées par ces virus. Les celluls hôtes expriment la protéine tronquée sur leur membrane extérieure et sécrètent cette protéine dans le milieu. L'invention décrit également des vaccins utilisables contre les infections par le flavivirus, ces vaccins contenant un virus de la vaccine recombinant, exprimant la protéine d'enveloppe tronquée de la présente invention, et/ou la protéine d'enveloppe tronquée produite par un baculovirus recombinant.
PCT/US1991/006031 1990-08-27 1991-08-27 Proteines d'enveloppe de flavivirus avec pouvoir immunogene accru utilisables dans l'immunisation contre les infections virales WO1992003161A1 (fr)

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EP0585549A3 (en) * 1992-06-11 1994-07-06 Nat Inst Health Method for producing ectoprotein of hepatitis c virus
EP0691404A2 (fr) 1994-07-08 1996-01-10 IMMUNO Aktiengesellschaft Vaccins améliorés pour l'immunisation contre l'encéphalite virale de la tique (TBE-virus) et méthode de fabrication
WO1996040933A1 (fr) * 1995-06-07 1996-12-19 The Government Of The United States Of America, Represented By The Secretary Department Of Health And Human Services Virus pdk-53 infectieux de la dengue 2 utilise comme vaccin quadrivalent
FR2741077A1 (fr) * 1995-11-14 1997-05-16 Pasteur Institut Vaccin polyvalent anti-dengue
WO1999006068A3 (fr) * 1997-07-31 1999-05-14 Hawaii Biotech Group Vaccin a enveloppe dimere recombinee contre les infections flavivirales
EP0836482A4 (fr) * 1995-05-24 1999-09-01 Hawaii Biotech Group Vaccin purifie contre une infection par flavivirus
WO2000012128A3 (fr) * 1998-08-28 2000-07-13 Hawaii Biotech Group Vaccin a sous-unite proteique non structurale recombinante contre les infections a flavivirus
US6676936B1 (en) 1988-07-14 2004-01-13 The United States Of America As Represented By The Department Of Health And Human Services. Chimeric and/or growth-restricted flaviviruses

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JOURNAL OF VIROLOGY, Vol. 64, issued September 1990, FALGOUT et al., "Immunization of mice with Recombinant Vaccinia Virus comprising Authentic Dergue Virus Non Structural protein NS1 protects against lethal Dergue Virus Encephalitis", pages 4356-4363. *
JOURNAL OF VIROLOGY, Vol. 64, No. 6, issued June 1990, YOSUDA et al., "Induction of Protective immunity in Animals Vaccinated with Recombinant Vaccinia Viruses that Express PreM and E Glycoproteins of Japanese Encephalitis Virus", pages 2788-2795. *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6676936B1 (en) 1988-07-14 2004-01-13 The United States Of America As Represented By The Department Of Health And Human Services. Chimeric and/or growth-restricted flaviviruses
US5830691A (en) * 1992-06-11 1998-11-03 Japan As Respresented By The Director General Of The Agency Of National Institute Of Health Method of producing ectoprotein of hepatitis C virus
EP0585549A3 (en) * 1992-06-11 1994-07-06 Nat Inst Health Method for producing ectoprotein of hepatitis c virus
EP0691404A2 (fr) 1994-07-08 1996-01-10 IMMUNO Aktiengesellschaft Vaccins améliorés pour l'immunisation contre l'encéphalite virale de la tique (TBE-virus) et méthode de fabrication
EP0836482A4 (fr) * 1995-05-24 1999-09-01 Hawaii Biotech Group Vaccin purifie contre une infection par flavivirus
WO1996040933A1 (fr) * 1995-06-07 1996-12-19 The Government Of The United States Of America, Represented By The Secretary Department Of Health And Human Services Virus pdk-53 infectieux de la dengue 2 utilise comme vaccin quadrivalent
FR2741077A1 (fr) * 1995-11-14 1997-05-16 Pasteur Institut Vaccin polyvalent anti-dengue
WO1997018311A1 (fr) * 1995-11-14 1997-05-22 Institut Pasteur Vaccin polyvalent anti-dengue
AU718740B2 (en) * 1995-11-14 2000-04-20 Institut Pasteur Polyvalent anti-dengue vaccine
WO1999006068A3 (fr) * 1997-07-31 1999-05-14 Hawaii Biotech Group Vaccin a enveloppe dimere recombinee contre les infections flavivirales
US6749857B1 (en) 1997-07-31 2004-06-15 Hawaii Biotechnology Group, Inc. Recombinant dimeric envelope vaccine against flaviviral infection
US6416763B1 (en) 1998-08-28 2002-07-09 Hawaii Biotechnology Group, Inc. Recombinant nonstructural protein subunit vaccine against flaviviral infection
WO2000012128A3 (fr) * 1998-08-28 2000-07-13 Hawaii Biotech Group Vaccin a sous-unite proteique non structurale recombinante contre les infections a flavivirus

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