WO1996040933A1 - Virus pdk-53 infectieux de la dengue 2 utilise comme vaccin quadrivalent - Google Patents

Virus pdk-53 infectieux de la dengue 2 utilise comme vaccin quadrivalent Download PDF

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WO1996040933A1
WO1996040933A1 PCT/US1996/009209 US9609209W WO9640933A1 WO 1996040933 A1 WO1996040933 A1 WO 1996040933A1 US 9609209 W US9609209 W US 9609209W WO 9640933 A1 WO9640933 A1 WO 9640933A1
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den
virus
leu
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thr
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PCT/US1996/009209
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Natth Bhamarapravati
Siritorn Butrapet
Jeffrey Chang
Duane J. Gubler
Scott B. Halstead
Richard Kinney
Dennis W. Trent
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The Government Of The United States Of America, Represented By The Secretary Department Of Health And Human Services
Mahidol University At Salaya
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Priority to AU60932/96A priority Critical patent/AU6093296A/en
Publication of WO1996040933A1 publication Critical patent/WO1996040933A1/fr

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    • 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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • 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
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    • 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
    • C12N2770/24121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N2770/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2770/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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 invention relates to infectious cDNA clones for Dengue 2 virus, strain 16681, and its live, attenuated vaccine derivative, PDK-53 (DEN-2 PDK-53) .
  • the invention also relates to infectious cDNA clones for chimeric viruses characterized as expressing structural genes of a Dengue 1, Dengue 3, or Dengue 4 attenuated virus in the context of the nonstructural genes of the Dengue 2 PDK-53 virus (DEN-2/1, DEN-2/3, DEN-2/4) .
  • the invention further relates to genetic constructs encoding these cDNAs, and host cells containing these constructs.
  • the invention moreover relates to quadravalent vaccines providing immunity against all four serotypes of dengue virus comprising DEN-2 PDK-53 infectious clone derivative, DEN- 2/1, DEN-2/3, or DEN-2/4 viruses, and related methods of immunization.
  • Arthropod-borne viruses are a diverse group of viruses that have been lumped together on the basis of their ecological niche, which involves cycles of transmission between vertebrate hosts and arthropod vectors such as mosquitos and ticks.
  • the prototype arbovirus is yellow fever virus, a flavivirus, which was isolated in 1927. In the 1950s, the Rockefeller
  • dengue hemorrhagic fever is one of the most important and increasing mosquito-transmitted infections in the world, with more than 85 countries in Asia, the Pacific Islands, Africa, Central America, and South America being threatened with dengue outbreaks.
  • Dengue fever was known in the past as "breakbone fever" due to the severe muscular and joint pain that accompanied the high fever during this infection. Dengue is an under-reported disease: it is thought that millions of cases occur each year.
  • Dengue (DEN) viruses which are flaviviruses, are classified antigenically into 4 serotypes (DEN-1, DEN-2, DEN-3, and DEN-4) . Multiple serotypes are now endemic in most countries in the tropics. DEN viruses are transmitted to humans principally by Aedes aegypti mosquitos throughout much of the tropical and subtropical region of the world. Viruses of all four serotypes infect humans and cause clinically inapparent infection or illness ranging from dengue fever to severe and often fatal dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) .
  • DHF/DSS dengue hemorrhagic fever/dengue shock syndrome
  • DHF/DSS has been associated epidemiologically and experimentally with immune enhancement of virus replication by preexisting, subneutralizing levels of heterotypic antibody. About 90% or more of patients with DHF/DSS are children who are 14 years old or younger (Halstead, 1970; Halstead, 1988) . Case fatality rates in untreated individuals can be as high as 15-20%. Between 1956 and 1978, hospitalization of more than 350,000 dengue patients and about 12,000 deaths in Southeast Asia were reported to the WHO (Halstead, 1980) .
  • Flaviviruses are enveloped RNA viruses 45 to 50 nm in diameter that contain a single-stranded, positive-sense capped RNA genome of approximately 11 kb. The RNA genome does not have a 3'-terminal poly(A) tail. Because the genetic molecule of flaviviruses is positive or messenger RNA (mRNA) -sense, naked genomic RNA injected, transfected, or electroporated into mammalian or invertebrate cells is capable of associating directly with the ribosomal protein synthetic machinery of the cell. All of the viral proteins are translated from the inserted viral genomic mRNA.
  • mRNA messenger RNA
  • the gene organization of the flavivirus mRNA genome is 5' -noncoding region (5' -NC) -capsid- premembrane/membrane (prM/M) -envelope (E) -nonstructural protein 1 (NS1) -NS2A-NS2B-NS3-NS4A-NS4B-NS5-3 ' -noncoding region (3'-NC).
  • 5' -NC 5' -noncoding region
  • prM/M premembrane/membrane
  • E envelopee
  • NS1 nonstructural protein 1
  • the structural proteins capsid, prM/M, and E and nonstructural proteins are translated as a large precursor polyprotein molecule from a single long open reading frame in the mRNA genome.
  • the individual mature viral proteins are processed from the polyprotein by both cell and virus specified proteases (Westaway et al . , 1985; Coia et al . , 1988; Speight and Westaway, 1989; Rice et al . , 1985 ) .
  • the structural proteins are those viral proteins that are incorporated into the mature virion.
  • the virion consists of an icosahedral capsid (C) that packages the viral genomic mRNA (nucleocapsid) .
  • the nucleocapsid is surrounded by a cell-derived lipid membrane into which the envelope (E) and mature membrane (M) proteins are imbedded.
  • the virus-specific nonstructural genes, NS1- NS5 are expressed in the cytoplasm of the infected cell and are involved in the replication and maturation of the viral RNA genome and viral proteins.
  • the E glycoprotein of the virus is exposed to the environment and is involved in attachment and entry of the virus into the cell.
  • the E protein is the primary viral immunogen against which the infected vertebrate host develops virus-specific neutralizing antibody.
  • the E gene is the most common target for development of molecular systems to express the encoded E glycoprotein.
  • immunization with various purified nonstructural genes of the virus have been shown to elicit protective immunity against challenge with wild-type virus, probably via cytotoxic T-cell mediated lysis of infected cells which express viral nonstructural proteins on the cell surface.
  • Vaccination can be one of the most cost effective ways to prevent dengue fever and DHF/DSS. Since 1979 the WHO has supported research on dengue vaccine development at the Mahidol University in Bangkok, Thailand (Press Release WHO/74, November 24, 1992) . Investigators at Mahidol University have developed four live, attenuated candidate vaccine viruses, one for each of the four serotypes, by serial passage of the virulent parent viruses in primary dog kidney (PDK) or fetal rhesus lung (FRhL) cell culture (Yoksan et al . , 1986; Bhamarapravati et al . , 1987). Phase 1 and Phase 2 clinical trials in Thailand have demonstrated that the vaccine is both safe and immunogenic in humans.
  • PDK primary dog kidney
  • FhL fetal rhesus lung
  • the vaccines now need to be tested for efficacy in large numbers of children (Press Release WHO/74, November 24, 1992) .
  • the invention provides a quadravalent vaccine providing immunity against all four serotypes of dengue virus comprising a DEN-2 PDK-53 infectious clone-derived virus.
  • the invention also provides a quadravalent vaccine providing immunity against all four serotypes of dengue virus comprising a chimeric DEN-2/1 virus.
  • the invention further provides a quadravalent vaccine providing immunity against all four serotypes of dengue virus comprising a chimeric DEN-2/3 virus.
  • the invention moreover provides a quadravalent vaccine providing immunity against all four serotypes of dengue virus comprising a chimeric DEN-2/4 virus.
  • the invention additionally provides a quadravalent vaccine providing immunity against all four serotypes of dengue virus comprising DEN-2 PDK-53 infectious clone-derived and chimeric DEN-2/1, DEN-2/3, and DEN-2/4 viruses.
  • the invention provides a method of immunization in which a desired immune response is produced against all four serotypes of dengue virus comprising the step of administering to a subject a quadravalent vaccine comprising DEN-2 PDK-53 infectious clone-derived and chimeric DEN-2/1, DEN-2/3, and DEN-2/4 viruses.
  • the invention provides a composition of matter comprising a full genome-length infectious cDNA clone for a DEN-2 virus, strain 16681.
  • the invention also provides a composition of matter comprising a full genome-length infectious cDNA clone for a DEN-2 virus of a strain characterized as replicating to high titer in cell culture.
  • the invention further provides a composition of matter comprising a full genome-length infectious cDNA clone for a DEN-2 virus, strain 16681, having the identifying characteristics of ATCC 69826.
  • the invention provides a composition of matter comprising a full genome-length infectious cDNA clone for a DEN-2 virus, strain 16681, attenuated derivative, PDK-53.
  • the invention also provides a composition of matter comprising a full genome-length infectious cDNA clone for a DEN-2 virus attenuated derivative, characterized as replicating to high titer in cell culture.
  • the invention further provides a composition of matter comprising a full genome-length infectious cDNA clone for a DEN-2 virus, strain 16681, attenuated derivative, PDK-53, having the identifying characteristics Of ATCC 69825.
  • the invention provides a composition of matter comprising a full genome-length infectious cDNA clone of a chimeric DEN-2/1 virus, wherein the virus is characterized as expressing the prM and E genes of a DEN-1 attenuated virus in the context of the nonstructural genes of the DEN-2 PDK-53 virus.
  • the DEN-1 attenuated virus may be DEN-1 PDK-13.
  • the invention also provides a composition of matter comprising a full genome-length infectious cDNA clone of a chimeric DEN-2 virus, wherein the virus is characterized as expressing the antigenicity of a DEN-1 attenuated virus.
  • the invention provides a composition of matter comprising a full genome-length infectious cDNA clone of a chimeric DEN-2/3 virus, wherein the virus is characterized as expressing the prM and E genes of a DEN-3 attenuated virus in the context of the nonstructural genes of the DEN-2 PDK-53 virus.
  • the DEN-3 attenuated virus may be DEN-3 PGMK30/FRhL-3.
  • the invention also provides a composition of matter comprising a full genome-length infectious cDNA clone of a chimeric DEN-2 virus, wherein the virus is characterized as expressing the antigenicity of a DEN-3 attenuated virus.
  • the invention provides a composition of matter comprising a full genome-length infectious cDNA clone of a chimeric DEN-2/4 virus, wherein the virus is characterized as expressing the prM and E genes of a DEN-4 attenuated virus in the context of the nonstructural genes of the DEN-2 PDK-53 virus.
  • the DEN-4 attenuated virus may be DEN-4 PDK-48.
  • the invention also provides a composition of matter comprising a full genome-length infectious cDNA clone of a chimeric DEN-2 virus, wherein the virus is characterized as expressing the antigenicity of a DEN-4 attenuated virus.
  • the invention provides a genetic construct comprising a DNA sequence operably encoding the polyprotein of DEN-2 virus, strain 16681.
  • the polyprotein may be the polyprotein encoded by the nucleotide sequence of SEQ ID NO:l.
  • the invention also provides a genetic construct comprising a DNA sequence operably encoding at least one protein of DEN-2 virus, strain 16681.
  • the protein may be a protein encoded by the nucleotide sequence of SEQ ID NO: 1.
  • the invention provides a genetic construct comprising a DNA sequence operably encoding the polyprotein of DEN-2 virus, strain 16681, attenuated derivative, PDK-53.
  • the polyprotein may be the polyprotein encoded by the nucleotide sequence of SEQ ID NO:2.
  • the invention also provides a genetic construct comprising a DNA sequence operably encoding at least one protein of DEN-2 virus, strain 16681, attenuated derivative, PDK-53.
  • the protein may be a protein encoded by the nucleotide sequence of SEQ ID NO: 2.
  • the invention provides a genetic construct comprising a DNA sequence operably encoding at least one structural protein of DEN-1 PDK-13.
  • the structural protein may be a structural protein encoded by the nucleotide sequence of SEQ ID NO: 124.
  • the invention provides a genetic construct comprising a DNA sequence operably encoding at least one structural protein of DEN-3 PGMK30/FRhL-3.
  • the structural protein may be a structural protein encoded by the nucleotide sequence of SEQ ID NO: 125.
  • the invention provides a genetic construct comprising a DNA sequence operably encoding at least one structural protein of DEN-4 PDK-48.
  • the structural protein may be a structural protein encoded by the nucleotide sequence of SEQ ID NO: 126.
  • the invention includes a host cell comprising any of the above genetic constructs.
  • Figure 1 Strategy for construction of the full genome-length cDNA clone of DEN-2 virus.
  • cDNA is amplified from the genomic RNA of the virus and cloned. Subclones are spliced together at unique, overlapping restriction enzyme sites to construct the full genome-length clone. Numbered arrows upstream (right arrows) and downstream (left primers used to amplify the cDNA in PCR reactions.
  • Figure 2 Transcription of genomic mRNA from the full-length infectious cDNA clone of DEN-2 virus.
  • the recombinant plasmid is linearized at the unique Xbal site at the 3 ' -end of the genomic cDNA.
  • Bacteriophage T7 RNA polymerase recognizes the T7 promoter engineered at the 5 '-end of the cDNA and transcribes full-length viral mRNA from the cDNA template.
  • Figure 3 Restriction enzyme sites identified in the nucleotide sequence of the RNA genome of DEN-2 16681 virus. Locations for the sites are indicated by the genome nucleotide numbers. Restriction enzymes that cleave the DEN-2 genomic cDNA at only a single location are listed vertically at the top of the figure. The resolution of the RENZ graph is 97.5 nucleotides per dot.
  • Figure 4 Growth curve of DEN-2 16681 virus in C6/36 mosquito cells.
  • Figure 5 (A) Polaroid prints showing RT/PCR amplification of the entire mRNA genome of DEN-2 virus, strain 16681, in the form of 5 cDNA amplicons.
  • the molecular weight marker (MW) consists of linear, double- stranded DNA markers of various base pair (bp) lengths.
  • the top 2 gels show 5- ⁇ l aliquots of the original RT/PCR reactions.
  • the bottom two gels show 10% of the yield following HMC agarose gel purification of the remaining 95- ⁇ l reaction aliquots.
  • B Primers (amplimers) used in the RT/PCR reactions and the expected sizes of the resulting cDNA amplicons.
  • FIG. 6 EcoRI restriction enzyme digests of F2, F2-Sal, Sal-F2, and F3 miniprep recombinant plasmid DNA. Plasmids from individual colonies resulting from transformation with independent ligated, recombinant plasmid molecules are numbered. The insert in the single F2-8 plasmid was too small and was discarded. The remaining recombinant plasmids contained cDNA inserts of expected size. As expected, F2-Sal cDNA contained two internal EcoRI sites; the Sal-F2 and F3 plasmids contained a single internal EcoRI site. EcoRI digestion of the recombinant plasmids regenerated linearized, wild-type 3.9-kb pCRII vector. For an undetermined reason, one of the EcoRI sites in plasmid F3-1 did not cut.
  • Figure 7 Schematic diagram showing the genomic locations of DEN-2 16681 virus-specific cDNA clones.
  • Clones indicated with asterisks were spliced together at the indicated restriction enzyme sites to construct the full genome-length cDNA clone.
  • Black horizontal bars indicate clone regions that were sequenced.
  • Light gray regions of horizontal bars indicate clone regions that were not sequenced.
  • Figure 8 (A) Effect of adding Taq extender reagent to PCR reactions. The 5.2-kbp amplicon of St. Louis encephalitis virus was readily obtained by extended PCR (+) but not by standard PCR (-) . (B) Agarose gel electropherogram showing DEN-2 PDK-53 Fl, F2, and F3 amplicons derived by extended PCR.
  • Figure 9 Schematic diagram showing the genome locations of errors identified in the cDNA clones of DEN-2 16681. Errors are indicated by short vertical tick marks.
  • Figure 10 Schematic diagram illustrating the approximate genome locations of the nucleotide discrepancies between the data of Applicants and those of Blok et al. (1992) for the sequence of the genome of DEN-2 virus, strain 16681.
  • Figure 11 Nucleotide sequence of the genome of DEN- 2 strain 16681 virus. Differences between the data determined by Blok e ⁇ al . (1992) (DEN-2-16681.BLOK) and those obtained by Applicants (DEN-2-16681.RK) . The genome nucleotide positions of the sequence differences are listed vertically. The solid squares indicate those nucleotide differences that also encode amino acid substitutions. The remaining nucleotide differences are either silent, encoding the same amino acid, or lie within the 5 ' -noncoding (5'-NC) or 3 ' -noncoding region (3 '-NO .
  • Figure 12 Schematic diagram showing the DEN-2 PDK- 53 virus-specific cDNA clones and the approximate locations of cDNA errors (vertical tick marks) identified by nucleotide sequence analyses. Clones marked with an asterisk were used in the construction of the DEN-2 PDK-53 virus-specific full-length cDNA clone. Clone #19 had a 203-bp deletion (horizontal line) .
  • Figure 13 Schematic summary of the DEN-2 16681 vs. PDK-53 virus sequencing projects. Arrows indicate the nucleotide differences detected between the two genomes. Triangles indicate those nucleotide changes that resulted in amino acid substitutions.
  • Figure 14 Finalized nucleotide and amino acid sequence of the RNA genome of DEN-2 virus, strain 16681 (SEQ ID N0:1) .
  • the EcoRI, SstI, Mull,and T7 promoter sites that were engineered immediately preceding the 5 ' - terminal nucleotide of the virus-specific genomic cDNA are shown.
  • the start positions of the viral genes and noncoding regions (5'-NC and 3 ' -NC) are shown.
  • Figure 15 Construction of intermediate clone F2 by ligating the F2-Sal Sphl/Hpal fragment and Sal-F2 Hpal/Kpnl fragment into pUCl ⁇ . The resulting F2 clone contained a nonsilent cDNA error at genome nucleotide position 1730.
  • Figure 16 Correction of the intermediate F2 clone. A new PCR amplicon was cloned and sequenced. The Sphl/Hpal fragment of this clone was spliced into F2 to construct F2-C having the correct nucleotide at genome position 1730.
  • Figure 17 Construction of the intermediate Fl/3/4/5 cDNA clone for DEN-2 16681 virus.
  • the thick solid black bars indicate DEN-2 virus-specific cDNA, illustrated with the RENZ sites of the MCS of the plasmid.
  • the RENZ sites used in each step of the splicing strategy are indicated in underlined, bold characters.
  • the top half of the figure shows construction of Fl/3/4/5-pUC18.
  • the bottom portion of the figure illustrates the making of Fl/3/4/5- pUC19.
  • the final step in the construction of the full genome-length cDNA clone involved the ligation of the F2-C Sphl/KpnI cDNA fragment into plasmid containing cDNA
  • Fl/3/4/5 and cut with RENZs Sphl/KpnI Although F2-C cDNA could not be cloned into Fl/3/4/5-pUC18, it was readily cloned into Fl/3/4/5-pUC19.
  • the pUC18 plasmid containing a small insert of cDNA made for Venezuelan equine encephalitis (VEE) virus was used simply to move Fl and F4/5 into pUC18 in a 3-molecule ligation reaction.
  • the VEE virus-specific cDNA was spliced out during this process. Arrowheads under cDNA bars indicate orientation of mRNA-sense cDNA strand.
  • Figure 18 Orientation specific cloning of full genome-length cDNA of DEN-2 16681 virus into the multiple cloning site of pUC19. Although the full-length cDNA was readily cloned in pUC19, multiple attempts to insert the cDNA into pUC18 failed. Presumably, interaction of the cDNA with pUC18-specific gene transcripts, translation of a toxic DEN-2 polypeptide, or translation of a toxic pUC18/DEN-2 fusion polypeptide produced deleterious effects in E. coli . Large arrows indicate orientation of mRNA-sense cDNA strands in the pUC plasmid backbone.
  • pUCl ⁇ EcoRI (BL) /Hindlll MCS fragment was ligated into pBR322 cut with Aval (BL) /Hindlll to make pBRUC-139.
  • BL pBRUC-138
  • pBRUC-139 3022-bp (61-bp MCS + 2961-bp pBR322 deletion vector) . Orientations of ORI, ROP, and the Amp gene are indicated.
  • Figure 20 Construction of pD2/IC-30P, the full genome-length cDNA clone of DEN-2 16681 virus, in plasmid pBR322 (pBRUC-139 (SphI-) derivative) .
  • the F3/4/5 clone cDNA was ligated into pBRUC-139 first (Top of Figure) , followed by Fl-E and F2-C.
  • Viable, infectious DEN-2 virus was successfully obtained from viral mRNA transcribed from this clone.
  • Figure 21 Construction of pD2/IC-130V, the full genome-length cDNA clone of DEN-2 PDK-53 virus.
  • a nonsilent error in cDNA clone F3-3C was corrected by splicing in a correct BstBI/Nhel fragment from clone F3.5- 6 (Top) .
  • the resulting corrected clone F3-3CC was spliced into the 16681 F345-F clone in pBRUC-139.
  • cDNA fragments F1-79B, F2-16B, and the recombinant F3/4/5 vector DNA were spliced together in a single ligation reaction to produce pD2/IC-130V.
  • the Nhel site occurs at genome nucleotide position 6646. Therefore, the PDK-53 virus-specific full- length cDNA clone contains the parental 16681 virus- specific nucleotide at position 8571. This nucleotide difference is silent; it does not encode an amino acid change. Other than the 8571 position, DEN-2 16681 and PDK-53 viruses are identical in nucleotide sequence from nucleotide position 6646 to the 3' terminus of the genome.
  • FIG 22 Agarose gel electropherogram of viral genomic mRNA extracted from gradient-purified, wild-type DEN-2 16681 virus and Venezuelan equine encephalitis (VEE) virus.
  • the quantity of RNA loaded onto the gel ranged from 22 ng to 383 ng.
  • the stock RNA was quantitated spectrophotometrically at 260 nm.
  • the genome-length RNA band is clearly visible between the 4153-bp and 6788-bp MW marker bands. Bands were visualized by incorporating 200 ng/ml of ethidium bromide stain in the gel and electrophoresis buffer.
  • Figure 23 Transcription of RNA from pVE/IC-92 (VEE virus clone) and pD2/IC-20 (DEN-2 16681 virus clone) .
  • Transcription reaction conditions 100 ng linearized DNA template, 12.5 mM DTT, 2.7 u/ ⁇ l RNasin, 0.15 mM NTPs, 3.3 U/ ⁇ l T7 RNA polymerase (Stratagene) in commercial buffer (Stratagene) ) yielded high quantity and quality of infectious mRNA transcripts from the pVE/IC-92 clone and 3 ' -end truncation products of that clone.
  • Figure 24 Transcription of RNA from the DEN-2 16681 cDNA clone pD2/IC-20.
  • A Transcription of RNA using different quantities of linearized plasmid template (a,b) .
  • the cap analog m7G(5 ' )ppp(5 ' )A was not included in the reaction.
  • B Transcription of 5 '-capped RNA with inclusion of cap analog in the reaction. Transcription was accomplished with the Ampliscribe transcription kit from Epicentre Technologies.
  • T7 pol bacteriophage T7 RNA polymerase.
  • Figure 25 Transcription of full genome-length, infectious viral mRNA from Xbal-linearized DEN-2 16681 plasmid pD2/IC-30P (A and D replicate clones resulting from independent bacterial colonies transformed with the recombinant pBRUC/DEN-2 plasmid) and PDK-53 plasmid pD2/IC-130V (F and J replicates) .
  • Genomic "viral RNA" extracted from gradient-purified wild-type DEN-2 16681 virus was electrophoresed in lanes 2 and 10. Aliquots of transcription reactions sampled before (T7 RNA polymerase "-”) and after (T7 Pol "+”) addition of T7 RNA polymerase are shown. Only the linearized plasmid DNA template is observed in the absence of the polymerase.
  • Figure 26 Transcription of RNA from pD2/IC-20, pD2/IC-30P, and pD2/IC-130V in the presence or absence of T7 RNA polymerase or cap analog in the transcription reaction. All lanes shown are on a single gel. Transcription was performed with the Ampliscribe transcription kit.
  • Figure 27 Derivation tree for the construction of the DEN-2 16681 and PDK-53 virus-specific full genome- length cDNA clones pD2/IC-30P and pD2/IC-130V, respectively, and chimeric 16681/PDK-53 clones derived from the two prototype clones.
  • Figure 28 Genotype maps of DEN-2 16681 and PDK-53 virus-specific full genome-length cDNAs and their chimeric derivatives. The scale at the top indicates relative genome nucleotide position in thousands. The graph resolution is 119.1444 bp/dot. cDNA regions contributed by the parental DEN-2 16681 virus are indicated by solid black bars. Regions derived from the DEN-2 PDK-53 vaccine virus are indicated by stippled bars. The 8 mutations identified by sequence analyses of the genomes of the 16681 and PDK-53 viruses are indicated. The virus- specific 5-noncoding nucleotides are indicated in lower case characters. The amino acids encoded by the virus- specific nucleotide mutations in the protein coding region of the genome are indicated in upper case, single-letter amino acid abbreviation.
  • Figure 29 Results of spot-sequencing PCR amplicons amplified from seed stocks of viruses derived from full genome-length cDNA clones. Dots indicate nucleotide sequence identity to the DEN-2 16681 virus. The expected virus-specific nucleotides for the genotype of each virus are shown. Those nucleotide positions that have actually been confirmed by sequence analysis are indicated by underlined nucleotide base characters. The actual genome nucleotide positions are indicated at the bottom of the Figure.
  • Figure 30 Recombinant full-length pD2/IC-30P-A and pD2/IC-130V-F plasmids extracted from 1-ml aliquots of E. coli TB-1 cultures submitted to ATCC.
  • Figure 31 Partial nucleotide sequences of candidate vaccine viruses:
  • DEN-1 16007 PDK-13 (Dl.VAC) (SEQ ID NO: 124)
  • DEN-2 16681 PDK-53 (D2.VAC) ( ⁇ e_e_ SEQ ID NO: 2) DEN-3 16562 PGMK-30/FRhL-3 (D3.VAC) (SEQ ID NO: 125) DEN-4 1036 PDK-48 (D4.VAC) (SEQ ID NO: 126) aligned with the nucleotide and deduced amino acid sequences of DEN-2 16681 virus (see SEQ ID NO:l) . Dots in the DEN-1, DEN-3, and DEN-4 sequences signify identity with the DEN-2 sequence.
  • Figure 32 Partial amino acid sequences of candidate vaccine viruses:
  • DEN-1 16007 PDK-13 (Dl.VAC) (SEQ ID NO: 124)
  • DEN-2 16681 PDK-53 (D2.VAC) ( ⁇ £e_ SEQ ID NO: 2)
  • DEN-3 16562 PGMK-30/FRhL-3 (D3.VAC) (SEQ ID NO: 125)
  • DEN-4 1036 PDK-48 (D4.VAC) (SEQ ID NO: 126) aligned with the deduced amino acid sequence of DEN-2 16681 virus (£££ SEQ ID NO:l) .
  • Dots in the DEN-1, DEN-3, and DEN-4 sequences signify identity with the DEN-2 sequence.
  • Figure 33 Mutagenesis analysis of the 5' end of the prM gene.
  • the 447-452 sequence (“AACCAC” in DEN-2) can be mutated to "CTCGAG” in all four DEN viruses to create a Xhol site for cassette splicing. This modification results in conservative Thr-Thr to Ser-Ser substitutions at amino acid positions prM 4-5 in DEN-2 virus. By creating this Xhol site, all four viruses will contain the sequence FHLSSR at amino acid positions prM 1-6 (see Figure 32) .
  • Nucleotide mutations that are necessary to create the Xhol site are indicated by bold, underlined characters in the nucleotide sequences of D2.VAC, Dl.VAC, D3.VAC, and D4.VAC and their respective primers designed for amplification in PCR.
  • FIG 34 Mutagenesis analysis of the 3' end of the E gene.
  • the 2344-2349 sequence (“TCACGC” in DEN-2) can be mutated to "TCTAGA” in all four DEN viruses to create a Xbal site for cassette splicing. This modification results in no amino acid change in DEN-2 at this site, but substitutions do occur in the other three viruses. By creating this Xhol site, all four viruses will contain the sequence SRS at amino acid positions E 470-472 (see Figure 32) .
  • Nucleotide mutations that are necessary to create the Xbal site are indicated by bold, underlined characters in the nucleotide sequences of D2.VAC, Dl.VAC, D3.VAC, and D4.VAC and their respective primers designed for amplification in PCR.
  • Figure 35 Construction of DEN-2 PDK-53 cassette plasmids pFl-Xho and pF2-Xba.
  • pFl-Xho Clone PCR cDNA amplicons Fl-prM5' and Fl-prM3' into TA-vector. Sequence and splice correct clones together at the SphI site in the TA-vector to ' construct pFl-prM53 (not shown) . Subclone the prM53 cDNA into Sstl/Sphl-cut pFl-E (see Figure 20) to construct pFl-Xho.
  • Figure 36 Construction of chimeric plasmids containing the prM and E genes (Xhol-Xbal cDNA fragment) of DEN-1, DEN-3, or DEN-4 candidate vaccine virus within the genetic background of DEN-2 PDK-53 virus.
  • pD2V-CAS12 was constructed by ligating the Sstl/SphI fragment of pFl- Xho and Sphl/KpnI fragment of pF2-Xba (see Figure 33) into a truncated form of pD2/IC-130V (see Figure 21) .
  • pD2/IC- 130V was truncated by restricting the full-length clone at the NsiI-4696 and 3 ' -end Xbal sites, blunt-ending with T4 DNA polymerase, and religating. This procedure removed genome nucleotides 4696-10723, thereby removing the Xhol- 5426 and 3 ' -end Xbal sites, which would otherwise interfere with construction of chimeric plasmid cassettes using Xhol and Xbal sites.
  • the cassette strategy employs PCR amplification of DEN-1, DEN-3, and DEN-4 cDNAs containing the prM and E genes; cutting the amplicons with Xhol/Xbal; cloning resulting fragments into pD2V-CAS12 to construct pDlV-CAS12, pD3V-CAS12, and pD4V-CAS12 chimeric cassettes; confirming the chimeric Xhol/Xbal insert by nucleotide sequence analysis; and then subcloning the Sstl/Kpnl fragment of the chimeric cassette into pD2/IC- 130V to construct the chimeric full genome-length cDNA clones from which chimeric DEN-2/1, -2/3, and -2/4 viruses are derived.
  • the genetic background of DEN-2 PDK-53 virus is illustrated by the solid black bars.
  • the heterologous DEN-1, DEN-3, and DEN-4 cDNA inserts are indicated by the stippled bars.
  • the infectious clone strategy was initiated with the virulent parental 16681 strain obtained from the Division of Vector-Borne Infectious Diseases (DVBID) of the Centers for Disease Control and Prevention (CDC) virus collection.
  • DVD Vector-Borne Infectious Diseases
  • CDC Centers for Disease Control and Prevention
  • the first full-length sequence-characterized cDNA clone was constructed in the high copy number pUC19 plasmid vector.
  • Successful transcription of genome-length DEN-2 16681 viral RNA from pD2/IC-20 was clearly demonstrated by agarose gel electrophoresis of the transcription reaction product.
  • RNA transcribed from this particular clone failed to yield infectious virus. It was determined that cDNA errors had occurred during the clone manipulations.
  • the full-length cDNA of DEN-2 16681 virus was successfully moved into pBR322 to construct pD2/lC-30P.
  • Full-length, infectious DEN-2 16681 genomic RNA was subsequently transcribed from pD2/IC-30P.
  • the DEN-1 PDK-13, DEN-2 PDK-53, DEN-3 PGMK-30/FRhL-3, and DEN-4 PDK-48 vaccine viruses were obtained from Mahidol University. Our goal involved replacement of the entire genomic cDNA backbone of the DEN-2 16681 full- length clone with the cognate cDNA cloned from the genome of the DEN-2 PDK-53 candidate vaccine virus. The prM and E genes of the DEN-2 PDK-53 virus are then replaced with the prM and E genes of the DEN-1 PDK-13, DEN-3
  • DEN-2 PDK-53 Infectious cDNA Clone Backbone c M E NS1 2A 2B NS3 4A 4B NS5 3'-NC
  • chimeric, infectious clone- derived DEN-2/1, DEN-2/3, and DEN-2/4 viruses will result in immediate improvement in the efficacy of a quadravalent vaccine.
  • Our preliminary data from Mahidol University indicate that very small amounts of the DEN-2 PDK-53 vaccine virus were required to infect and immunize humans.
  • the DEN-1, DEN-3, and DEN-4 vaccine virus candidates had approximately 30-fold to 2000-fold lower infectivity for humans.
  • the low infective efficacies of the DEN-1, DEN-3, and DEN-4 viruses create significant problems in terms of vaccine efficacy in eliciting seroconversion in vaccinees, as well as problems of vaccine production for mass vaccination programs, since a large volume, up to 1 ml, of undiluted cell culture- derived vaccine virus must be administered to achieve even minimal levels of infectivity for these viruses.
  • chimeric vaccine viruses that express the relevant immunogenic structural proteins of DEN-1, DEN-3, or DEN-4 virus in the context of replication control by the nonstructural gene products of the DEN-2 PDK-53 virus should replicate better and be more infective and immunogenic in human vaccinees than the original DEN-1, DEN-3, and DEN-4 vaccine viruses containing nonchimeric genotypes.
  • a quadravalent vaccine is obtained upon completion of the following steps:
  • a full genome-length infectious cDNA clone for a DEN2-16681 attenuated derivative, PDK-53 is constructed, preferably by substituting the genomic cDNA backbone of the DEN2-16681 full length clone with the corresponding cDNA cloned from the genome of the DEN-2 PDK-53 candidate vaccine virus.
  • the candidate DEN-1, DEN-3, and DEN-4 vaccine viruses are subjected to PCR amplification of cDNA from extracted genomic RNA, and chimeric infectious cDNA clones expressing the prM and E genes of DEN-1, DEN-3, and DEN-4 viruses, respectively, in the context of the nonstructural genes of the DEN-2 PDK-53 virus are constructed.
  • the infectious clone-derived chimeric DEN-2/1, DEN-2/3, and DEN-2/4 vaccine viruses are tested to ensure that they:
  • DEN viruses are naturally transmitted between mosquitos and humans. Although lower primates can be infected with these viruses, they do not develop the clinical profiles that occur in humans. Infectious clone-derived viruses can be compared to their more virulent parental strains using certain in vi tro and in vivo markers:
  • CPE Cytopathic effects
  • Virulence by intracranial route in mice Virulence by intracranial route in mice; Viremia in monkeys; Virulence by intracranial route in monkeys; and
  • RNA transcripts are transfected into permissible cells, and the live, attenuated viruses are formulated into vaccines.
  • the DEN-2 PDK-53 and chimeric DEN- 2/1, DEN-2/3, and DEN-2/4 infectious cDNA clones can by themselves confer immunity by DNA immunization, a form of gene therapy involving the direct inoculation of naked DNA into the host such that its expression produces an immune response (e.g., Ulmer et al . , 1993 (DNA immunization protected against influenza); Cox et al . , 1993 (DNA immunization protected against herpesvirus); Xiang et al . , 1994 (DNA immunization protected against rabies) ; Sedegah et al . , 1994 (DNA immunization protected against malaria) ) .
  • infectious cDNA clones are extraordinaries for studying the molecular biology of virus structure, function, and replication. This has been amply demonstrated for many RNA viruses in the literature, including Venezuelan equine encephalitis virus as reported by Kinney et al . (1989).
  • a successful infectious cDNA clone of DEN-2 virus permits important investigations of dengue virus replication, pathogenesis, and antigenic structure.
  • In ectious clone cDNA templates permit the directed engineering of virus vaccines. Directed site- specific, nonrandom mutations can readily be made in infectious cDNA clones, and therefore in clone-derived viruses, using a wide variety of DNA modification enzymes, restriction endonucleases, and in vi tro mutagenesis methods.
  • RNA DNA is easier to manipulate than RNA, and the 10" 9 error rate of DNA replication is much lower than the 10 ⁇ 3 - 10' 4 error rate produced by RNA polymerases.
  • Infectious cDNA clones permit direct analyses of the phenotypic effects of individual and cumulative mutations in the viral genome.
  • An infectious cDNA clone provides a "gold standard" reference sequence for a vaccine.
  • the virulent parental DEN-2 16681 strain was immediately available in the DVBID collection of viruses.
  • the DEN vaccine viruses were passaged in primary dog kidney (PDK) cells because this cell culture is included among those cell types that are certified for human use by the Bureau of Biologies, US Food and Drug Administration (Yoksan et al . , 1986) .
  • the virus strain designations are shown below:
  • PDK primary dog kidney cells
  • FRhL fetal rhesus lung cells
  • PGMK primary green monkey kidney cells
  • the DEN-2 full-length cDNA clone was derived from the DVBID seed of DEN-2 16681 virus, which had the passage history:
  • cDNA Complementary DNA
  • Stock virus seed was prepared from virus-infected cells grown in 75 or 150 cm 2 plastic tissue culture flasks.
  • the culture medium was clarified by centrifugation for 30 min at 10,000 rpm in a Sorvall GSA rotor, bringing the final concentration of fetal bovine serum (FBS) to 10% (v/v) , and then freezing the clarified virus suspension in aliquots of 0.5 - 1.0 ml at -70°C.
  • Gradient purified DEN- 2 16681 virus was prepared according to the method of Obijeski et al . (1976) as reported by Kinney et al . (1983) .
  • Infectious virus was derived from the infectious cDNA clones by electroporation of BHK-21-15 (baby hamster kidney-21, clone 15) cells with transcribed viral RNA. Viruses were also grown in LLC-MK 2 monkey kidney cells, Vero African green monkey kidney cells, and C6/36 mosquito cells (Aedes albopictus C6 cells, clone 36, Igarashi (1978)) .
  • All four cell lines were grown in Eagle's minimal essential medium (MEM) supplemented with 10% (v/v) heat-inactivated (56°C for 30 min) FBS, 1.25 g/L of sodium bicarbonate, 100 units/ml of penicillin G, and 100 ⁇ g/ml of streptomycin sulfate.
  • Confluent cell monolayers grown in plastic tissue culture flasks were infected by decanting the growth medium, permitting the virus inoculum to adsorb for 1.5 h at 37°C, and then adding MEM containing 5% FBS.
  • MEM minimal essential medium
  • confluent cell monolayers in plastic 6-well trays were inoculated with 200 ⁇ l of the appropriate dilution of virus.
  • Virus was adsorbed to the cell monolayer for 1.5 h at 37 °C.
  • the cells were then overlaid with 3 ml of 1% (w/v) Noble agar (maintained at 40°C) in MEM lacking phenol red pH indicator and containing 2% FBS and 0.01% (w/v) DEAE-dextran.
  • a second 1-ml agar overlay containing 50 ⁇ g/ml of neutral red vital stain was added. Viral plaques were counted 2-5 days later.
  • the E. coli K-12 strains used in this project included XLl-Blue, MC-1061, SURE, JM101, and TB-1.
  • Recombinant plasmid containing full genome-length cDNA of DEN-2 virus was successfully replicated in E. coli XLl- Blue, MC-1061, and TB-1. Flavivirus cDNA, particularly the gene region encoding the envelope glycoprotein, is troublesome in E. coli .
  • Bacteria hosting the recombinant plasmid containing the full-length cDNA clone grew slowly and were often difficult to streak for isolation on agar plates containing selective antibiotic. Transformation efficiencies were sometimes improved somewhat by incubation of agar plates at 30°C or ambient temperature rather than at 37°C. Bacterial stocks were stored frozen at -70°C in 10% (v/v) glycerol.
  • RNA is a fragile molecule that is very readily degraded by the many ubiquitous RNases present in the environment. Many of these RNases are resistant to treatment with detergents and heat, including autoclaving. All reagents and materials that contacted the viral RNA in this project were RNase-free to avoid degradation of the viral RNA by these ubiquitous, very stable enzymes.
  • the investigator wore tight-fitting gloves, maintained all reagents on ice, used a plastic tool to open the lids of microtubes, used individually packaged pipets, preferably plastic for aqueous solutions, disposable plasticware which is generally RNase-free before opening, and used "For RNA Only" microtubes, Gilson micropipetors (P-10, P- 20, P-100, P-200, P-1000) and tips with aerosol barriers. Use of recycled glassware was avoided. Weigh boats, magnetic stirrers, and pH meters were not used. Chemicals were weighed in sterile, RNase-free disposable plastic 50- ml centrifuge tubes, and solutions were adjusted to the appropriate pH by aliquoting a small volume of the solution onto pH paper.
  • Virus seeds containing at least 10 6 PFU (plaque forming units) /ml of virus are ideal for providing appropriate yields of RNA. Seed with virus titer of 10 4 or lower can be problematic in terms of yielding sufficient RNA. For these low-titer seeds it is best to pool the yields of several extracted seed aliquots.
  • RNA extraction involved the addition of 200 ⁇ l of cold RNA lysis buffer (4 M guanidine isothiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% (w/v) sarkosyl, and 100 mM beta-mercaptoethanol) , and 30 ⁇ l of 3 M sodium acetate, pH 5.2, to an empty RNase-free 1.5-ml microtube on ice.
  • 200 ⁇ l of DEN virus seed was added to the microtube and mixed vigorously for 30 sec with a mechanical mixer.
  • the tube was centrifuged briefly to pellet the liquid; then 400 ⁇ l of cold phenol (commercially supplied by AMRESCO) equilibrated to pH 4.5 and 80 ⁇ l of cold chloroform were added.
  • the tube was mixed vigorously for 30 sec, placed on ice for 15 min, mixed again, then centrifuged for 1 min at maximum speed in a refrigerated microcentrifuge to separate the aqueous and organic phases.
  • the top aqueous phase containing the extracted RNA was transferred to a fresh 1.5-ml microtube on ice, 400 ⁇ l of cold isopropanol was added, and the tube was incubated for at least 1 h or overnight at -20°C.
  • RNA was precipitated by centrifugation for 10 min at maximum speed at 4°C. The supernatant was removed with a pipet rather than by decantation and rinsed with 500 ⁇ l of 75% (v/v) ethanol. After spinning again for 10 min, the ethanol was removed with a pipet . The tube was centrifuged again briefly and the residual liquid was removed with a micropipet. The RNA pellet was air dried briefly, resuspended in 50 ⁇ l of cold RNase-free dH 2 0, and stored frozen. For seeds containing low virus titer, the RNA pellets in 3-6 microtubes were pooled in a total volume of 50 ⁇ l.
  • RT/PCR reverse transcriptase/poly erase chain reaction
  • thermocyder The RT/PCR reactions in thin-wall 200- ⁇ l microtubes (Phenix Research Products) were incubated without oil overlay in a Perkin-Elmer Model 9600 thermocyder according to the following program:
  • HMC High-Melt-Crush
  • agarose gel slice containing DNA was placed in a 1.5-ml microtube and crushed thoroughly with a spatula or pestle.
  • the volume of the crushed agarose was brought to 400-500 ⁇ l with TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM disodium EDTA) and 400 ⁇ l of phenol (supplied by AMRESCO) , pH 8, was added.
  • TE buffer 10 mM Tris-HCl, pH 7.5, 1 mM disodium EDTA
  • phenol supplied by AMRESCO
  • the top aqueous phase was transferred to a fresh microtube, extracted with 400 ⁇ l of phenol:chloroform:isoamyl alcohol (25:24:1) and centrifuged for 2 min.
  • the top aqueous phase was transferred to a fresh tube and extracted with 700 ⁇ l of diethyl ether or chloroform. If chloroform was used, the top phase was again transferred to a fresh tube after a brief spin to separate phases.
  • the DNA was precipitated for at least 30 min at -70°C or overnight at -20°C following addition of 2.5 volumes (essentially filling the microtube) of 95% ethanol containing 300 mM ammonium acetate and 10 mM MgCl 2 .
  • the DNA was pelleted at 4°C by centrifugation for 20 min at maximum speed. The liquid was decanted, and the DNA pellet was rinsed with 500 ⁇ l of 75% ethanol, air-dried briefly, dissolved in 30 ⁇ l of TE buffer, and stored frozen or in the refrigerator. A 3- ⁇ l aliquot of the extracted DNA was analyzed for purity and quantity by agarose gel electrophoresis. Generally,
  • DNA was analyzed by electrophoresis in 1% (w/v) agarose gels run in TBE buffer (100 mM Tris-HCl, pH 8, 91 mM boric acid, and 20 mM disodium EDTA) . DNA bands were visualized by staining the gel in water containing 500 ng/ml of ethidium bromide and exposure to ultraviolet light. Gels used for analyzing RNA transcripts were made with RNase-free reagents. Ethidium bromide stain was incorporated in the gel and running buffer so that the RNA bands could be visualized immediately.
  • DNA was electrophoresed in 0.7% (w/v) agarose gels made with genetic technology grade Seakem agarose (FMC) or with biotechnology grade agarose (3:1 high resolution blend, AMRESCO) .
  • TA- cloning vectors have been engineered (Marchuk et al . , 1991) . These vectors generally have a single "T” overhang engineered at the 3 ' -terminus of EcoRV-cut, blunt-ended, linearized plasmid vector. The EcoRV site occurs within the multiple cloning site (MCS) of the plasmid.
  • MCS multiple cloning site
  • the MCS is a series of contiguous, unique restriction enzyme (RENZ) sites engineered into a vector plasmid to permit subcloning of exogenous DNA fragments following restriction with a variety of RENZs.
  • RENZ contiguous, unique restriction enzyme
  • the HMC-purified DEN cDNA amplicons were cloned into the 3900-bp pCRII (Invitrogen) , the 2887-bp pT7Blue (R) (pT7Blue, Novagen) , or the 3003-bp pGEM-5Zf (Promega) TA-vector plasmid.
  • RENZ sites available in the MCS region of these TA- vectors as well as the RENZ sites of the MCS of the general purpose cloning plasmids, pUCl ⁇ and pUC19, used in this project are shown below.
  • the pUC18/19 plasmids possess identical MCS sites in reverse orientation in the plasmid backbone. Their purpose is to permit cloning of DNA in either orientation into the plasmid using the same pair of RENZs - this reversibility was exploited in this project.
  • the TA- vectors used here all possessed T7 and/or SP6 bacteriophage RNA promoters to enable RNA transcription from cloned DNA. These promoters were not used in this project. All of the plasmids contain the gene for ampicillin resistance. They also contained the lac Z portion of the E. coli lac operon. This permits color discrimination between bacterial colonies that receive a recombinant or a wild-type plasmid.
  • Agar plates contained 800 ⁇ g of IPTG and 800 ⁇ g of X-gal.
  • HMC-purified amplicon Fifty to 100 ng of HMC-purified amplicon was ligated to 50 ng of the pCRII vector using the TA-vector cloning kit supplied by Invitrogen exactly as specified by the instructions supplied with the kit.
  • the transformed cells were plated on YTA 50 agar plates (8 g of DIFCO tryptone, 5 g of DIFCO yeast extract, 5 g of NaCl, and 15 g of BACTO agar per liter of dH 2 0) containing 50 ⁇ g/ml of ampicillin.
  • Electroporation-competent cells were prepared by growing a fresh bacterial culture to an optical density of 0.5-0.7 at 600 nm. The cells from 1.5 - 3 L of culture were pelleted by centrifugation for 10 min at 4°C and 5000 rmp in a Sorvall GSA rotor, pooled, washed twice in 1 mM Hepes buffer, and resuspended in 2 ml of 10% (v/v) sterile glycerol per L of original culture. The concentrated cells in glycerol were stored at -70°C.
  • Cloning amplicons larger than 3500 bp into the TA-vector can be very difficult.
  • miniprep plasmids were selected for further analysis. Their corresponding bacterial minicultures were streaked for isolation on YTA 50 plates, and an isolated colony was inoculated into 50-200 ml of YTA 50 broth to grow up a preparative amount of recombinant plasmid.
  • the preparative scale for the extraction of the plasmid was essentially identical to that for minipreps except for scaled up volumes.
  • the supernatant was aspirated, and the cell pellet was resuspended gently by up/down micropipeting in 200 ⁇ l of GTE buffer (50 mM glucose, 25 mM Tris-HCl, pH 8.0, and 25 mM disodium EDTA) and then mixed with 300 ⁇ l of lysis buffer (0.2 N NaOH, 1% (w/v) sodium dodecylsulfate (SDS)) . After incubation on ice for 5 min, 300 ⁇ l of cold potassium acetate solution (3 M potassium acetate, 7 M acetic acid, pH 4.8) was added, and the solution was chilled for 5 min on ice and then centrifuged at maximum speed for 10 min at 4°C.
  • GTE buffer 50 mM glucose, 25 mM Tris-HCl, pH 8.0, and 25 mM disodium EDTA
  • 300 ⁇ l of lysis buffer 0.2 N NaOH, 1% (w/v) sodium dodecylsulfate (
  • the supernatant was poured into a fresh microtube, RNase A was added to 20 ⁇ g/ml, and the mixture was incubated at 37°C for 30 min.
  • the sample was extracted twice with 600 ⁇ l of chloroform and centrifuged for 1 min at maximum speed at room temperature.
  • the DNA pellet was dissolved in 32 ⁇ l of dH 2 0.
  • Eight ⁇ l of 4M NaCl and 40 ⁇ l of 13% (w/v) PEG-8000 was added, and the mixed solution was incubated for 5 min on ice.
  • the sample was centrifuged for 15 min at maximum speed at 4°C, the liquid was aspirated with a micropipet , and the pellet was rinsed with 500 ⁇ l of 75% ethanol.
  • the air dried pellet was dissolved in 30 ⁇ l of dH 2 0 and stored frozen until used.
  • Preparative-scale plasmid extraction was performed by inoculating 100 ml of 2X-YTA 50 broth with 2 ml of an overnight culture of E. coli . The culture was shaken overnight at 300 rpm and 37°C. The cells were pelleted by centrifugation for 10 min at 5000 rpm in a Sorvall GSA rotor and resuspended in 6 ml of cold GTE buffer. Nine ml of a freshly made solution of 0.2 N NaOH and 1% (w/v) SDS was added. The sample was incubated for 5 min on ice, then 9 ml of cold 3 M potassium acetate solution was added.
  • the tube was centrifuged for 20 min at 10,000 rpm at room temperature and the supernatant was transferred to a fresh 30-ml glass tube.
  • RNase A was added to 20 ⁇ g/ml, and the sample was incubated for 30 min at 37°C and then extracted twice with 6 ml of chloroform. Twelve ml of room- temperature isopropanol was added and the tube was centrifuged immediately for 20 min at 10,000 rpm at room temperature. The supernatant was decanted, and the DNA pellet was rinsed with 1 ml of 75% ethanol, air dried briefly, and resuspended in 480 ⁇ l of dH 2 0.
  • the DNA was precipitated by addition of 120 ⁇ l of 4 M NaCl and 600 ⁇ l of 13% PEG-8000, incubation for 5 min on ice, and centrifugation for 15 min at maximum speed at 4°C.
  • the DNA pellet was rinsed with 500 ⁇ l of 75% ethanol, air dried briefly, rehydrated in TE buffer, and stored frozen.
  • Nucleotide sequence analyses of DEN-2 16681 cDNA clones #1-#15 were performed by cloning EcoRI restriction fragments of each clone into the single-stranded bacteriophage M13mpl8 or M13mpl9. Since this is not the current method of choice for sequencing, the method will be described only briefly here.
  • the procedure used for the extraction of plasmid DNA from bacterial cells was also used to extract the intracellular double-stranded replicative form (RF) DNA of M13 from bacteriophage- infected J57. coli JM101 cells.
  • the RF DNA was linearized at the EcoRI site of the MCS and ligated to the DEN-2 HMC- purified EcoRI cDNA restriction fragments.
  • Electroporation-competent E. coli JM101 cells were transformed by electroporation and plated onto H-agar plates (10 g of DIFCO tryptone, 5 g of NaCl, 15 g of BACTO agar, and 1% (w/v) thiamine per liter of dH 2 0) containing 800 ⁇ g each of isopropyl- ⁇ -D-galactopyranoside (IPTG) and 5-bromo-4-chloro-3-indolyl- ⁇ -D-galactopyranoside (BCIG or X-gal) .
  • H-agar plates 10 g of DIFCO tryptone, 5 g of NaCl, 15 g of BACTO agar, and 1% (w/v) thiamine per liter of dH 2 0
  • IPTG isopropyl- ⁇ -D-galactopyranoside
  • BCIG or X-gal 5-bromo-4-chloro-3-ind
  • the electroporated cells were mixed with 300 ⁇ l of a fresh logarithmic culture of JM101 cells and 3 ml of warm (51°C) top H-agar containing 9 g/L of agar and then poured onto the H-agar plates.
  • Cells that were transfected with recombinant DNA supported replication of recombinant M13 virus, resulting in the formation of bacteriophage plaques in the JM101 cell lawn on the agar plate.
  • the IPTG/BCIG histochemistry of the system permitted identification of white plaques containing recombinant bacteriophage into which cDNA had been ligated into the EcoRI site of the MCS, whereas wild-type nonrecombinant M13 bacteriophage produced blue plaques .
  • Isolated plaques were picked, inoculated into 3 ml of a fresh, pre-logarithmic phase culture of JM101, and shaken at 37°C for 8-16 h.
  • the minicultures were clarified by centrifugation in 1.5-ml microtubes, the bacteriophage particles were precipitated with PEG-8000, and the single- stranded, circular bacteriophage DNA was isolated from the virions by phenol extraction.
  • the recombinant, circular, single-stranded bacteriophage DNA was sequenced by the dideoxynucleotide termination method. Sequencing kits can be purchased from various commercial vendors. Radioactive 32 P-dCTP or 35 S-dCTP was incorporated into the strands synthesized in the sequencing reactions. Sequencing was accomplished with many DEN-2 virus-specific primers designed to sequence the entire genome. The sequence reactions were electro-phoresed in 6% (w/v) polyacrylamide gels, which were dried onto filter paper and overlaid with X-ray film. The DNA bands of the autoradiographs were read by the investigator, and the data was entered into a sequence project data spreadsheet. This sequencing method has been used extensively in the past ( e . g.
  • Nucleotide sequencing was also performed by the current method of direct sequencing of double-stranded plasmid DNA by the dideoxynucleotide termination method using the Applied Biosystems Taq DyeDeoxy Terminator Cycle Sequencing Kit, cycle sequencing in the Model 9600 thermocyder according to the instruction manual supplied with the kit, and analyzing the DNA sequence on an ABI Model 373A DNA sequencing apparatus.
  • Sequencing reactions in 200- ⁇ l thin-walled microtubes contained 9.5 ⁇ l of reaction mix (buffer, the four dideoxynuleotides, and Taq polymerase supplied in the kit) , 7.0 ⁇ l of double or single-stranded template DNA (150 pg/bp) , and 3.2 ⁇ l of 10 ⁇ M sequencing primer (32 pmol) . After mixing, the reactions were placed in a Perkin-Elmer Model 9600 thermocyder, and programmed cycle sequencing was performed for 25 cycles of incubation at 96°C for 15 sec, 50°C for 15 sec, and 60 C C for 4 min.
  • Strand extension was performed at 60°C rather than 72°C because the fluorescent dye-labeled dideoxynucleotide terminators are heat sensitive.
  • the reaction was then applied to a Centrisep gel column (Princeton Separations) to remove unincorporated dye-labeled dideoxynucleotides according to the instructions supplied with the columns.
  • the eluted DNA was vacuum dried for 1 h using a Savant Speed Vac Concentrator and stored at -70°C.
  • the DNA was hydrated with 5 ⁇ l of deionized formamide and 1 ⁇ l of 50 mM disodium EDTA, then heated in an aluminum block for 2 min at 90°C.
  • a 3- ⁇ l aliquot of the denatured DNA sample was applied to one of 24 wells of a polyacrylamide-urea gel in an Applied Biosystems 373A DNA sequencer.
  • the color-coded sequence chromatograph was read by visual inspection, and the resulting nucleotide sequence was entered into a computer-maintained sequence data spreadsheet.
  • the sequencing kit incorporates dideoxynucleotide terminators that are each labeled with a unique fluorescent dye that permits laser detection of all four terminators in a single polyacrylamide gel lane in the Model 373 sequencer.
  • the data was recorded in the form of colored chromatograms that are easily read by the investigator.
  • Single-stranded recombinant M13 DNA can also be sequenced in this manner.
  • White bacteriophage plaques containing recombinant M13 DNA were picked with sterile toothpicks and placed into 2-ml slightly turbid (less than 0.15 A 600 ) cultures of E. coli JM101. The cultures were shaken at 300 rmp and 37°C overnight and then clarified by centrifugation in microtubes at maximum speed for 10 min at room temperature. One ml of the supernatant was transferred to a fresh 1.5-ml microtube containing 200 ⁇ l of sterile 20% (w/v) PEG-8000 in 250 mM NaCl. The tubes were mixed by inversion, incubated for 15 min at room temperature, and centrifuged at maximum speed for 5 min at room • temperature.
  • the PEG supernatant was removed completely, and the DNA pellet was resuspended in 300 ⁇ l of TE buffer.
  • An equal volume of pH 8-buffered phenol was added, and the solution was mixed vigorously several times during a period of 20 min at room-temperature.
  • the tube was centrifuged for 5 min at room temperature, and the top aqueous phase was transferred to a fresh 1.5-ml microtube.
  • the DNA was precipitated by adding 2.5 volumes of 95% ethanol containing 300 mM ammonium acetate and 10 mM MgCl 2 and incubating at -20°C overnight.
  • the tube was centrifuged at maximum speed for 15 min at 4°C, and the supernatant was decanted. Following a rinse with 500 ⁇ l of 75% ethanol, the DNA was air dried briefly, resuspended in 60 ⁇ l of TE buffer, and stored at 4°C.
  • Primer design was based on the sequence of DEN-2 virus, strain 16681, published by Blok et al . (1992) , and DEN-2 virus, Jamaican strain 1409, as reported by Deubel et al . (1986) and Deubel et al . (1988) .
  • Primers were synthesized by the Biotechnology Core Facility at the CDC in Atlanta, Georgia. We received the dried primers via mail and adjusted them to a concentration of 100 ⁇ M in dH 2 0. The designations and sequences of all of the primers-amplimers used in this project are listed in Appendix A.
  • a downstream amplimer was designed that was complementary to the published sequence of the 3 ' terminus of the genome.
  • a unique Xbal restriction enzyme site was incorporated at the 5 ' end of this amplimer to provide a unique site to permit linearization of the recombinant plasmid containing the full-length cDNA clone at the 3' terminus of the cloned genomic cDNA. This linearization was necessary to obtain appropriately terminated DEN virus-specific run-off RNA transcripts from the cDNA clone in transcription reactions with bacteriophage T7 RNA polymerase.
  • Amplimer CD2-10687.XBA or CD2-10687.X2 was used to amplify and clone the 3 '-terminal portion of DEN-2 16681 or PDK-53 virus, respectively.
  • the promoter for the bacteriophage T7 RNA polymerase was engineered at the 5 ' terminus of the cloned genomic cDNA by incorporating the recognition sequence of the T7 RNA polymerase into the sequence of the 5 '-terminal upstream, mRNA-sense amplicon D2-SMT71 immediately preceding the 5 '-terminal nucleotide of the DEN-2 viral genome.
  • the recombinant plasmid containing the full-length cDNA clone was prepared for RNA transcription by linearization at the unique Xbal site located at the 3 ' terminus of the cloned genomic cDNA.
  • the restriction reaction containing the Xbal-restricted plasmid was extracted sequentially with phenol:chloroform:isoamyl alcohol and chloroform and then precipitated.
  • the DNA was redissolved in 50 ⁇ l of TE buffer and digested with proteinase K at a concentration of 1 mg/ml for 1 h at 37°C to hydrolyze contaminating RNases.
  • the sample was then extracted twice with "For RNA Only" phenol:chloroform:isoamyl alcohol buffered to pH 8, extracted twice with chloroform to remove traces of phenol, and precipitated by adding one-tenth volume of RNase-free 3 M sodium acetate, pH 5.2, and 2.5 volumes of ethanol and incubating for at least 1 h at -70°C or overnight at -20°C.
  • DEN-2 virus-specific genomic RNA was transcribed from the linearized cDNA template using a commercial T7 transcription kit (Ampliscribe T7 transcription kit, Epicentre Technologies) . Transcription reactions were performed for 2 h at 37°C in RNase-free 1.5-ml microtubes in 20- ⁇ l reactions containing 100-1000 ng of linearized DNA template, 7.5 mM each of CTP, GTP, and UTP, 0.75 mM ATP, 2.7 mM m 7 GpppA cap analog, 6.7 mM DTT, 2.0 ⁇ l of a 10X concentration of a proprietary buffer supplied with the commercial kit, and 2.0 ⁇ l of the proprietary Ampliscribe enzyme solution supplied with the kit. Reaction solutions were used directly and without further treatment to transfect BHK-21 cells.
  • Ampliscribe T7 transcription kit Epicentre Technologies
  • BHK-21 clone 15 cells were transfected with RNA transcripts by electroporation (Liljestr ⁇ m et al . , 1991) .
  • Fresh cultures of BHK-21 cells were grown to 90% confluency, rinsed twice with cold RNase-free phosphate buffered saline (PBS) , and released from the plastic by incubation with 3 ml of commercial trypsin-EDTA solution (GIBCO-BRL) .
  • the cells were pelleted by low-speed centrifugation at 1200 rpm for 5 min in a Beckman GPKR centrifuge. The cells were washed twice with cold PBS, resuspended in cold PBS and kept on ice.
  • the cells were counted using a hemacytometer and microscope, and the cell concentration was adjusted to 10 7 cells/ml. One-half ml of the washed, adjusted cells were mixed with each transcription reaction solution in 1.5-ml microtubes on ice. The mixture was transferred to a cold electroporation cuvette with 0.2-cm electrode gap, which was placed in the cuvette holder of the Bio-Rad Gene Pulser. The cells were shocked twice using settings of 1.5 kV voltage, 25 ⁇ FD of capacitance, and resistance set to infinity. The shocked cells were incubated for 10 min at room temperature and then added to 75 cm 2 tissue flasks containing 20 ml of MEM containing 10% FBS.
  • Transfected cell cultures were incubated at 37°C for 5-8 days until CPE was evident in the cell monolayer and/or expression of DEN virus-specific antigens was identified in an aliquot of the cell monolayer scraped from the flask using DEN virus-specific mouse hyperimmune ascitic fluid or monoclonal antibodies in indirect immunofluorescence tests.
  • DEN-2 16681 virus replicates to high titer in cell culture.
  • the CDC virus seed used in this study contained 2.0 X 10 7 plaque forming units (PFU) /ml. This titer was determined by plaque titration of the seed virus in monolayer cultures of Vero cells. This seed titered 1.3 X 10 4 PFU/ml in LLC-MK 2 cells. A growth curve for this virus was determined in C6/36 Aedes albopictus cell culture ( Figure 4) . This level of replication is quite high for a flavivirus.
  • the DEN-2 16681 virus is eminently suitable to serve as the parent to an infectious cDNA clone of DEN virus.
  • the DEN-2 PDK-53 vaccine virus taken directly from a vaccine vial obtained from Mahidol University, contained 3.4 X 10 4 PFU/ml of virus, as titrated in Vero cell monolayers, and 1.5 X 10 4 PFU/ml as titrated in LLC-MK 2 cell monolayers.
  • DEN-2 virus parental strain 16681
  • genomic RNA was amplified from genomic RNA in the form of 5 cDNA clones of various sizes (T7-F1, F2, F3, F4, and F5) .
  • PCR amplification with 5 sets of upstream and downstream amplimers yielded the predicted amplicon sizes in PCR reactions.
  • Figure 5 shows the migration of these cDNA fragments in agarose gels.
  • Recombinant plasmids obtained by ligating the cDNA amplicons into the pCRII TA-vector, were extracted from minicultures derived from transformed E. coli XLl-Blue colonies. Uncut plasmids were screened for the presence of cDNA insert by comparing their mobility in agarose gels with the mobility of uncut wild-type pCRII vector plasmid. Selected plasmids were then restricted with the restriction enzyme EcoRI to confirm the size of the inserted cDNA fragment. EcoRI digests of F2-Sal, Sal-F2, and F3 plasmids derived from independent transformed bacterial colonies are shown in Figure 6.
  • FIG. 8B shows the correct agarose gel migration of large cDNA amplicons Fl (containing the T7 RNA polymerase promoter at the 5' end of the mRNA-sense strand of the amplicon), F2, and F3 obtained by PCR amplification using DEN-2 PDK-53 viral genomic RNA as template.
  • the standard PCR reaction also worked for a number of DEN-2 PDK-53 amplifications.
  • the PDK-53 PCR products were cloned into the pGEM-5Zf TA-vector (Promega) or the pT7Blue(R) TA-vector (Novagen) . Although we seemed to have the best cloning efficiency of PCR amplicons in the pCRII TA-vector, the other vector kits were less expensive and worked well. The cloning efficiency of PCR products into the TA-vector decreased rapidly as amplicon size increased beyond 2000 bp.
  • RNA transcribed from this clone was not infectious.
  • One of these mutations was a base deletion in the NS4B gene. This deletion would cause a frameshift of the amino acid sequence, resulting in ribosomal translation of a nonsense polypeptide downstream of the mutation point.
  • nucleotide sequence of DEN-2 16681 virus that we determined at the CDC laboratory differed significantly from the sequence of DEN-2 16681 virus as published by Blok et al . (1992) .
  • Our nucleotide sequence differed from that published by Blok et al . (1992) at 60 nucleotide positions, which were located throughout the genome. Amino acid substitutions were encoded by 26 of these nucleotide differences.
  • the approximate genomic locations of the nucleotide differences are illustrated in the schematic diagram in Figure 10. The exact nucleotide positions of the discrepancies are shown in Figure 11.
  • the DEN-2 PDK-53 virus-specific cDNA clones were analyzed by direct sequencing of the double-stranded plasmid DNA by the thermocycling method using the Taq DyeDeoxy Terminator Cycle Sequencing Kit.
  • the 3 ' -end sequence from nucleotide position 10290-10686 was also determined by direct sequencing of PCR-derived amplicon cDNA. Sequence analysis was performed using the automated 373A DNA sequencing machine. The color-coded sequence chromatograms were read by the investigator and the data was entered manually into a computer-based spreadsheet.
  • the approximate locations of the cDNA errors identified in the PDK-53 cDNA clones are illustrated in Figure 12.
  • Table Summary of nucleotide differences between the genomes of DEN-2 16681 virus and its vaccine derivative virus, strain PDK-53.
  • our PDK-53 virus-specific cDNA clones did not result from contamination of PDK-53- specific PCR reactions with 16681 virus-specific cDNA template.
  • Our PDK-53 virus-specific cDNA clones which also contained the many sequence discrepancies between our data and those of Blok et al. (1992) , encoded the nucleotide sequence from the 5 ' terminus to nucleotide position 10337 of the genome of PDK-53 virus.
  • the 3'- terminal 387 nucleotides (10337-10723) of DEN-2 PDK-53 virus were identical to those of the parental 16681 virus.
  • clone #5 contained a cDNA "error" that was not readily spliced out with the existing clones. This error, which was a C-to-T mutation at nucleotide position 1730 and encoded a nonsilent Thr-to-lie amino acid substitution at E-265, was incorporated into the F2 construct.
  • the intermediate F2 construct was the result of splicing the F2-Sal clone (#5) Sphl/Hpal fragment to the Sal-F2 clone (#7) Hpal/Kpnl fragment in the MCS of plasmid pUC18 ( Figure 15) .
  • a new PCR amplicon was made using primers D2-1261 and CD2-2955. Resulting clones in the TA-vector were sequenced, and the correct Sphl/Hpal fragment of a new clone was substituted for the faulty Sphl/Hpal fragment of the original F2 construct ( Figure 16) .
  • the corrected F2 clone was designated F2-C.
  • the relevant cDNA clones of DEN-2 16681 virus were spliced together via a series of intermediate ligation products in the MCS of pUC18 to yield Fl/3/4/5, which contained all of the genome except for the Sphl-Kpnl 1380- 4493 region present in clone F2-C.
  • Multiple attempts to ligate the F2-C Sphl/KpnI cDNA fragment into Fl/3/4/5 in pUC18 failed.
  • the cDNA insert of Fl/3/4/5-pUC18 was then transferred to the MCS of pUC19, resulting in Fl/3/4/5- pUC19. This operation simply reversed the orientation of the cDNA insert within the context of the pUC plasmid.
  • the full genome-length cDNA of DEN-2 16681 virus was cloned into the MCS of pUC19.
  • Apparent full genome-length viral mRNA was transcribed from linearized pD2/IC-20. This transcribed product failed to yield infectious virus following electroporation of BHK-21 cells.
  • Most of the cDNA in the pD2/IC-20 clone was resequenced, and several cloning artifacts, including a fatal single-nucleotide deletion, were identified.
  • Original subunit intermediate cDNA constructs in pUC18 were resequenced to confirm that they possessed the correct sequence and corrected where necessary.
  • the corrected primary cDNA clones Fl, F2-C, and F3/4/5 were then ligated into the low-copy plasmid pBR322, rather than the high copy-number pUC18 plasmid. It was envisioned that the cDNA would be more stable in a slower-replicating plasmid in E. coli .
  • the MCS of pUC19 was spliced into the pBR322 plasmid ( Figure 19) .
  • the SphI site was removed from both pBRUC plasmids by cutting with SphI, blunt ending of the cut ends using T4 DNA polymerase, and then ligating the ends back together. This was necessary for the construction of the full-length cDNA clone because SphI is one of the cDNA restriction/splicing sites for the clone.
  • the full-length infectious clone of DEN-2 16681 virus was used in the construction of the infectious clone for PDK-53 virus. Since the 3 ' -noncoding regions of the genomes of both viruses are identical, and the amino acid sequences of the translated precursor polyproteins encoded by genome nucleotide positions 6646-10269 are identical in both viruses, the infectious clone of PDK-53 virus was constructed using the 16681 3 ' -end cDNA from the Nhel site at nucleotide position 6646 to the 3 ' terminus of the genome ( Figure 21) .
  • this fragment and the F2-16B cDNA fragment were ligated into the infectious clone backbone to construct the DEN-2 PDK-53 virus-specific full-length cDNA clone, pD2/IC-130V ( Figure 21) .
  • FIG. 22 shows an agarose gel electropherogram for 22-383 ng of viral genomic RNA obtained from purified preparations of wild-type DEN-2 16681 virus and wild-type Venezuelan equine encephalitis (VEE) virus, strain Trinidad donkey. Although degradation of the RNA is visible as a spectrum of smaller molecular weight nucleic acid (smear in Figure 22) , definite full-genome length RNA bands are clearly visible.
  • This smear of nucleic acid is probably also due, in part, to multiple conformations of the single-stranded RNA molecules which migrate through the gel at different rates.
  • the relative gel migration of the single-stranded RNA does not correlate directly with the sizes of the double-stranded molecular weight marker
  • RNA bands (MW, Figure 22) ; the VEE and DEN-2 viral genomes are 11,447 and 10,723 nucleotides in length, respectively.
  • BHK-21 and C6/36 cells were transfected successfully by electroporation with 2000, 500, 100, 10, 1, and 0.1 ng of viral genomic RNA extracted from purified VEE or DEN-2 16681 virus, as indicated by development of CPE, expression of viral proteins detected by indirect immunofluorescence tests using virus-specific antibody, and/or by plaque titration of infectious virus from the transfected-cell culture medium.
  • RNA quantities of 1 ng or less were essentially undetectable in the ethidium bromide-stained agarose gel system we used. Therefore, authentic RNA transcripts derived from full genome-length cDNA and visualized in agarose gel electropherograms of transcription reactions should be infectious for BHK-21 cells by electroporation.
  • FIG. 23 shows an agarose gel electropherogram that demonstrates successful transcription of RNA from the VEE clone, but not pD2/IC-20.
  • RNA transcription from the DEN-2 clone In an attempt to improve RNA transcription from the DEN-2 clone, commercial transcription kits were purchased. The Megascript transcription kit supplied by Ambion also failed to transcribe RNA from the DEN clone. However, the Ampliscribe kit obtained from Epicentre Technologies enabled efficient transcrip-tion of RNA from the DEN-2 clone ( Figure 24) .
  • the success of the Ampliscribe kit apparently was due to the high concentration of ribonucleotides and a very high, but proprietary, concentration of T7 RNA polymerase.
  • the RNA transcribed from pD2/IC-20 was not infectious.
  • viral mRNA transcribed from DEN-2 16681 clone pD2/2-IC30P and PDK-53 clone pD2/IC-130V was infectious ( Figure 25) .
  • Viral mRNA transcripts from both replicates of pD2/IC-30P (A and D) and pD2/IC-130V (F and J) were infectious, producing viable infectious virus in electroporated BHK-21 cells.
  • Figure 26 shows RNA transcripts from pD2/IC-20, pD2/IC-30P, and pD2/IC-130V.
  • TB-1 were successfully transformed with ligated recombinant plasmids containing full genome-length cDNA. Viable virus was derived from all of the indicated clones.
  • Viable prototype and chimeric viruses were derived from each of the clones indicated in Figure 28 by electroporation of BHK-21 cells with viral genome-length mRNA transcribed from linearized plasmids. Seed stocks of these viruses were prepared by centrifuge-clarification of the cell culture medium, adjustment of the FBS concentration to 10%, and freezing of seed aliquots at -70°C. Virus concentrations were determined by plaque titration of the virus seeds in monolayer cultures of Vero cells. The results of these virus titrations are shown in the following table.
  • D2/IC-130V-F 4.0 X 10 5 t V . , D F .
  • D2/IC-130V2-1 2.8 X 10 5 t V . . . .
  • a D2/IC-130V2-7 8.8 X 10" t V . .
  • D2/IC-321-N 7.6 X 105 t V .
  • D F . . D2/IC-323-B 7.2 X 10 5 . . . D F .
  • Genotype is designated in small case for the virus-specific 5 ' -noncoding nucleotide and in upper case single-letter amino acid abbreviation for amino acids encoded by virus -specific nucleotide mutations. Dots represent nucleotide or amino acid sequence identity with DEN-2 16681 virus. To establish the validity of the clone-derived chimeric viruses, relevant genomic cDNA fragments were amplified directly from seed viruses by PCR and spot- sequenced. The results are shown in Figure 29. This validation process is ongoing.
  • clone-derived chimeric viruses have yet to be spot-sequenced in a recipient clone-derived cDNA region to definitely establish the chimeric nature of the virus.
  • the recipient clone is the recombinant plasmid backbone into which a cDNA fragment, the insert fragment, from a heterologous donor clone is spliced. Where duplicate clone-derived viruses were obtained, both viruses of a given genotype were spot-sequenced, and both gave the same result, which is shown in Figure 29.
  • Patent deposits of the full genome-length cDNA clones of DEN-2 16681 and PDK-53 viruses were submitted to the American Type Culture Collection (ATCC) , Rockville, Maryland, U.S.A. Both pD2/IC-30P-A and pD2/IC-130V-F were grown overnight in E. coli TB-1 cells.
  • ATCC American Type Culture Collection
  • Six cryogenic vials containing 1 ml each of frozen cell culture in 10% glycerol were submitted by dry ice shipment. Prior to shipment, plasmid was extracted from a 1 ml aliquot of each virus-specific culture. The recombinant full-length plasmid was recovered from the cells as shown in Figure 30.
  • the pD2/IC-30P-A deposit with the ATCC was assigned accession number ATCC 69826, and the pD2/IC-130V-F deposit with the ATCC was assigned accession number ATCC 69825. Date of deposit was May 25, 1995.
  • the amplified cDNA molecules were sequenced directly, thus providing the sequence of the population of virions in the virus seed.
  • the amplified cDNA amplicons for the DEN-1, DEN-3, and DEN-4 vaccine viruses have all been cloned into the pGEM-5Zf TA-vector.
  • the cloned cDNA has not been analyzed by sequencing, since it will be necessary to rederive the cDNA amplicons by PCR to incorporate appropriate RENZ cleavage sites within the amplicon for splicing into the full-length cDNA backbone of DEN-2 PDK-53 virus.
  • the partial nucleotide sequences of the genomes of the DEN-1, DEN-3, and DEN-4 vaccine viruses were aligned with the DEN-2 PDK-53 sequence. All four sequences are aligned with the nucleotide sequence of DEN-2 16681 virus and its deduced amino acid sequence in Figure 31. The deduced amino acid sequences of the DEN viruses are aligned in Figure 32.
  • mutagenic primers containing the appropriate RENZ site are utilized in PCR reactions to synthesize new cDNA for the prM and E genes of all four viruses.
  • a DEN-2 PDK-53 virus-specific cDNA cassette plasmid, designated pD2V-CAS12, containing the genome region from the 5 ' terminus through nucleotide position 4696 is constructed via intermediate plasmid constructs pFl-Xho and pF2-Xba as illustrated in Figures 35 and 36.
  • the Xhol/Xbal cDNA fragments cut directly from DEN-1, DEN- 3, and DEN-4 virus-specific amplicons synthesized by PCR using the mutagenic primers are ligated into the pD2V- CAS12 cassette plasmid to create subclone chimeras.
  • the Sstl/Kpnl fragment of the resulting pDlV-CAS12, pD3V- CAS12, and pD4V-CAS12 cassettes are moved into pD2/lC-130V restricted with Sstl/Kpnl to create the chimeric full genome-length cDNA clones ( Figure 36) .
  • Infectious cDNA clones permit the directed engineering of viral genomes. Depending on their viability in terms of ability to replicate in cell culture, infectious clone-derived viruses can be modified by incorporating point mutations, multiple mutations, deletions, gene regions of related or heterologous viruses, or nonviral genes. Infectious cDNA clones have been developed for many RNA viruses, including flaviviruses DEN-4 (Lai et al. , 1991) , yellow fever (Rice et al., 1989), Kunjin (Khromykh and Westaway, 1994), Japanese encephalitis (Sumiyoshi et al. , 1992), and TBE (unpublished data) .
  • the purpose of engineering chimeric DEN vaccine viruses is to enhance the replicative ability and immunogenicity of the DEN-1, DEN-3, and DEN-4 vaccine viruses.
  • a primary assumption has been that the attenuated DEN-2 PDK-53 vaccine virus replicates to appropriate levels in cell culture. In fact, it does appear that the genome of DEN-2 PDK-53 virus is eminently suited to serve as the genetic backbone for chimeric viruses containing the prM and E genes of DEN-1, DEN-3, and DEN-4 vaccine viruses.
  • the DEN-2 PDK-53 virus and its infectious clone derivative viruses grow to approximately 10 7 PFU/ml in LLC-MK 2 cells, about as well as the DEN-2 16681 virus.
  • a second assumption is that the chimeric DEN viruses will be viable and the DEN-2 PDK-53 virus-specific replication machinery will significantly increase replication of the chimeric viruses in cell culture and increase their infectivity and immunogenicity in humans relative to the wild-type vaccine viruses.
  • the high degree of conservation of amino acid sequences among the polyproteins of the four DEN viruses should ensure that the chimeric viruses will be viable.
  • the level of replication attained by the chimeric DEN viruses is determined empirically, as was determined for the DEN-2 PDK-53 infectious clone derivative virus.
  • DEN-1 and DEN-2 structural protein antigens in the genetic background of DEN-4 virus were spliced much of the 5 ' -noncoding region, and the capsid, prM and E genes of DEN-1 or DEN-2 virus into the full-length cDNA clone of DEN-4 virus.
  • the near 3 ' -terminal splice site they chose in the E gene is very close to that proposed by us in our project.
  • These chimeric viruses replicated very slowly relative to the wild-type viruses. The authors attributed this slow replication to possible suboptimal gene expression, assembly, and/or maturation due to incompatibility of heterotypic genes or RNA packaging in the nucleocapsid.
  • DEN virus chimeras may be derived that are viable.
  • Thr-to-Ser amino acid substitutions near the amino terminus of the prM protein in DEN-2, DEN-2/1, DEN-2/3, and DEN-2/4 viruses resulting from mutagenesis to create the Xhol site of the cassettes should be conservative in nature and affect the phenotype of derived viruses minimally, if at all.
  • a unique Mlul site (ACGCGT) could be created via a single, silent A-to-G point mutation at nucleotide position 453 in the DEN-2 clone. The Mlul site immediately preceding the T7 promoter could easily be eliminated by cutting the clone with Mlul, blunt-ending, and religation.
  • the clone-derived DEN-2 and chimeric viruses would then have the prM amino-terminal sequence "FHLTTR."
  • the carboxyl-terminal 24 amino acids of the E glycoprotein of all of the infectious clone-derived viruses will be those of the DEN-2 PDK-53 virus. Therefore, the E protein of all of the chimeric viruses will have amino acid mutations in this region.
  • the carboxyl-terminal 39 amino acids of the DEN virus E protein comprise membrane-spanning, transmembrane domains. In all enveloped viruses, the transmembrane domains of the integral viral proteins of related viruses are quite variable in amino acid sequence.
  • the E protein of all flaviviruses share a similar gross tertiary structure that is indicated by the absolute conservation of the 6 Cys residues in the prM protein and in the 12 Cys residues in the ectodomain (the .region located on environment side of the viral lipid envelope) of the E protein of DEN, Japanese encephalitis, West Nile, Murray Valley encephalitis, St. Louis encephalitis, Kunjin, yellow fever, TBE, Langat, and Powasson flaviviruses (data not shown) . Cys residues are involved in intrachain Cys-Cys disulfide bonds that determine the overall structure of the protein.
  • DEN-2/1, DEN-2/3, and DEN-2/4 chimeric viruses are viable and to replicate more efficiently than the wild- type DEN-1, DEN-3, and DEN-4 vaccine viruses, respectively.
  • chimeric recombinants involving the genetic backbone of one flavivirus and the structural genes of a variety of different flaviviruses may also be viable, as has been demonstrated for DEN-4/TBE virus recombinants (Pictnev et al., 1993).
  • Such recombinant viruses offer the potential opportunity to engineer chimeric vaccine viruses for a number of flavivirus-associated diseases within the genetic background of a single flavivirus.
  • DEN-2 PDK-53 virus has attenuating mutations in the noncoding regions or in the nonstructural genes and/or that attenuating mutations occur in the prM/E region of the genomes of DEN- 1, DEN-3, and DEN-4 viruses.
  • Mutations in essentially any region of the viral genome may be capable of attenuating a virulent virus. This has been demonstrated for a number of viruses including polio virus, VEE virus, and Theiler's virus. Noncoding as well as protein coding regions may be involved in attenuation. Attenuating mutations in the envelope proteins of enveloped viruses are common (Barrett et al. , 1990) .
  • the nucleotide mutations in DEN-2 PDK-53 virus at genome nucleotide positions 57 (5 ' -noncoding region) , 524 (prM) , 2579 (NS1) , 4018 (NS2A) , and 6599 (NS4A) may be involved in attenuation of the virus.
  • prM amino acid mutation is the only mutation affecting virulence of the virus, the DEN-2 PDK-53 genetic background, within which the structural genes from heterologous viruses will be expressed, does itself possess genotypic markers of attenuation.
  • Nucleotide sequence analysis of expressed genes is essential.
  • the error rate in the original RT/PCR derived cDNA clones of DEN-2 16681 virus was 8.2 x 10" 4 , that is 1 cDNA error for every 1227 nucleotides of cloned, sequenced cDNA.
  • the error rate was calculated to be 3.9 x 10 ⁇ 4 or 1 error for every 2543 nucleotides of cloned, sequenced cDNA.
  • RNA polymerases and reverse transcriptase have no editing function. Incorrect nucleotides incorporated during strand elongation are not detected or removed before continuing.
  • the Taq DNA polymerase is also known to incorporate errors into PCR amplicons. Thus, at least 4-8 cDNA "errors" can be expected to occur in 10 kb of cloned cDNA.
  • a faulty structural gene cDNA clone of the virulent VEE Trinidad donkey (TRD) virus that was expressed in recombinant vaccinia virus was essentially authentic by monoclonal antibody analysis of expressed VEE virus-specific proteins and by protection of immunized mice from challenge with virulent VEE virus (Kinney et al. , 1988a; Kinney et al. , 1988b) .
  • incorporation of this cDNA clone into an infectious cDNA clone of VEE virus completely abrogated the virulence of the clone- derived virus, whereas the corrected cDNA fragment resulted in derivation of virulent virus (Kinney et al. , 1993) .
  • Nucleotides encoding cDNA errors will be confirmed on both cDNA strands, but will not be identified as errors unless the sequences of two or more independent cDNA clones covering the same region of the genome are sequenced.
  • the functional full-length clone of DEN-4 virus was obtained by repeated splicing of large new cDNA fragments into the full-length clone until a functional clone was obtained. The authors did not indicate that the newly cloned regions were characterized by nucleotide sequence analysis (Lai et al., 1991) . It is probable that the slowed replication of the DEN-4/1 and DEN-4/2 chimeric viruses relative to wild-type viruses reported by Bray et al.
  • infectious clones There are now only two classes of infectious clones developed for vaccine flaviviruses that have themselves been administered to humans : the infectious clone of yellow fever virus, vaccine strain 17D (Rice et al. , 1989; Hahn et al. , 1987; Rice et al. , 1985) , and the DEN-1, DEN- 2, DEN-3, and DEN-4 vaccine derivative infectious clones described herein. Both classes of infectious clones have the important advantage of being derived from vaccine viruses that have been tested for efficacy and safety in humans.
  • the yellow fever 17D virus vaccine has long been one of the most effective human vaccines developed; immunization with this virus provides lifelong immunity.
  • DEN virus In the case of DEN virus, it is essential that vaccines provide immunity against infection by all four serotypes of the virus.
  • DEN-1, DEN-2, DEN-3, and DEN-4 vaccine viruses have been developed at Mahidol University, Bangkok, Thailand. All four vaccine viruses have been tested in humans and have been demonstrated to be immunogenic and safe for human adults.
  • Replicating vaccines in the form of live, attenuated viruses offer distinct advantages in terms of immunogenic efficacy due to replicative amplification of viral antigens (antigenic mass) in the vaccinees and replication in appropriate target tissues. Inactivated or subunit antigens usually suffer from a lack of sufficient antigenic mass and subsequent failure to stimulate an effective immune response.
  • Immunization with the live, attenuated VEE TC-83 vaccine virus provided immunity against both parenteral challenge (immunity provided by circulating serum IgG antibody) and intranasal challenge (mucosal, IgA-base immunity) with virulent VEE virus. Furthermore, the level of immunity, as measured by titers of VEE virus-specific neutralizing antibody, were considerably higher in TC-83 virus- immunized mice and horses (the natural epidemic host for VEE virus) than in animals immunized with recombinant vaccinia/VEE virus (Kinney et al., 1988b; Bowen et al. , 1992) .
  • vaccinia/influenza A virus recombinants Similar results have been reported for vaccinia/influenza A virus recombinants in rodents (Smith et al. , 1986). Furthermore, a replicating vaccine virus provides the appropriate T-cell epitopes to stimulate cell-mediated immunity as well as humoral immunity. T- cell epitopes may be lacking in subunit vaccines. In short, vaccination with a safe live, attenuated vaccine virus provides the optimal immunization of a natural infection in terms of the type and level of immunity elicited and the repertoire of viral antigens involved in generating the immune response.
  • DEN viruses described herein as vaccine candidates, it is necessary to rederive the viruses by transfection of a cell line, such as primary dog kidney, certified for human use under conditions of good laboratory practice and management to ensure the avoidance of potential adventitious agents that might be present in uncertified cell lines.
  • a cell line such as primary dog kidney
  • the cDNA-derived viruses originate from candidate vaccine viruses that have undergone testing in humans, they require recertification by analysis for possible in vi tro phenotypic markers of attenuation and by safety testing in small animals and probably nonhuman primates. All investigative studies involving the pathogenesis of DEN virus are hampered by the unavailability of a suitable animal model. Certain in vi tro characteristics are apparently associated with attenuation of DEN viruses, but the only definitive test is vaccine trial in human volunteers.
  • Vaccine trails would presumably follow those of the original wild-type vaccine viruses developed at Mahidol University.
  • the protocol includes titration of the individual vaccine virus candidates in adult human volunteers to determine the minimal infectious/immunogenic dose for each virus. This is followed by immunization trials with different bivalent and trivalent combinations of vaccine virus. The final test is the quadravalent vaccine composed of appropriate doses of all four vaccine viruses. If the preliminary trials are successful, larger trials are scheduled, and the vaccine viruses are tested in children, who are the primary target for vaccine delivery.
  • a wild-type vaccine virus serves as the template for the clone construction.
  • Large cDNA fragments are amplified from the genomic mRNA by PCR using virus-specific primers and directly cloned into a TA-vector or into the MCS of a low-copy number plasmid following restriction of the amplicon cDNA.
  • the low-copy pBRUC-139 vector contains the MCS of pUC19 to permit convenient cloning of cDNA using a variety of RENZ sites. Other low-copy plasmids are available.
  • the bacteriophage T7 or SP6 promoter is usually engineered into the 5'-terminal mRNA-sense amplimer, and a unique RENZ site for linearization of the recombinant plasmid containing the full-length cDNA must be engineered into the 3-terminal complementary (negative) -sense amplimer. Exhaustive nucleotide analysis of the cDNA clones is desirable.
  • Bovine Herpesvirus 1 Immune Responses in Mice and Cattle Injected with Plasmid DNA. J. Virol. 67:5664.
  • Arboviruses Including Certain Other Viruses of Vertebrates. American Society of Tropical Medicine and Hygiene, San Antonio, Texas.
  • VEE Recombinant vaccinia virus/Venezuelan equine encephalitis
  • Attenuation of Venezuelan equine encephalitis virus strain TC-83 is encoded by the 5 ' -noncoding region and the E2 envelope glycoprotein. J. Virol. 67:1269- 1277.
  • RNA transcripts from Sindbis virus cDNA clones mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vi tro mutagenesis to generate defined mutants. J. Virol. 61:3809-3819.
  • Dengue virus vaccine development study on biological markers of uncloned dengue 1-4 viruses serially passaged in primary kidney cells. In Arbovirus Research in Australia. Proceedings of the Fourth
  • CD2-378 25/- 5'-TGCAGATCTGCGTCTCCTATTCAAG-3 '
  • CD2-616 26/- 5'-TTGCACCAACAGTCAATGTCTTCAGG-3 '
  • CD2-771 25/- 5'-ATGTTTCCAGGCCCCTTCTGATGAC-3 '
  • NAME Spratt, Gwendolyn D.
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE: (ix) FEATURE:
  • AAG GCG AAA AAC ACG CCT TTC AAT ATG CTG AAA CGC GAG AGA AAC CGC 162 Lys Ala Lys Asn Thr Pro Phe Asn Met Leu Lys Arg Glu Arg Asn Arg 10 15 20
  • CAG GGA CGA GGA CCA TTA AAA CTG TTC ATG GCC CTG GTG GCG TTC CTT 258 Gin Gly Arg Gly Pro Leu Lys Leu Phe Met Ala Leu Val Ala Phe Leu 40 45 50
  • GGC ATG ATC ATT ATG CTG ATT CCA ACA GTG ATG GCG TTC CAT TTA ACC 450 Gly Met lie He Met Leu He Pro Thr Val Met Ala Phe His Leu Thr 105 110 115
  • TTC CAA AGA GCC CTG ATT TTC ATC TTA CTG ACA GCT GTC ACT CCT TCA 930 Phe Gin Arg Ala Leu He Phe He Leu Leu Thr Ala Val Thr Pro Ser 265 270 275
  • AAA ACA GAA GCC AAA CAG CCT GCC ACC CTA AGG AAG TAC TGT ATA GAG 1122 Lys Thr Glu Ala Lys Gin Pro Ala Thr Leu Arg Lys Tyr Cys He Glu 330 335 340
  • GGC AAG GAA ATC AAA ATA ACA CCA CAG AGT TCC ATC ACA GAA GCA GAA 1458 Gly Lys Glu He Lys He Thr Pro Gin Ser Ser He Thr Glu Ala Glu 440 445 450
  • GGC CAA ATG TTT GAG ACA ACA ATG AGG GGG GCG AAG AGA ATG GCC ATT 2178 Gly Gin Met Phe Glu Thr Thr Met Arg Gly Ala Lys Arg Met Ala He 680 685 690
  • GAA TTT GCA GCC GGA AGA AAG TCT CTG ACC CTG AAC CTA ATC ACA GAA 6402 Glu Phe Ala Ala Gly Arg Lys Ser Leu Thr Leu Asn Leu He Thr Glu 2090 2095 2100
  • GCA CAT TAT GCC ATC ATA GGG CCA GGA CTC CAA GCA AAA GCA ACC AGA 7218 Ala His Tyr Ala He He Gly Pro Gly Leu Gin Ala Lys Ala Thr Arg 2360 2365 2370
  • GGC ATT AAA AGA GGA GAA ACG GAC CAT CAC GCT GTG TCG CGA GGC TCA 7746
  • GCA AAA CTG AGA TGG TTC GTT GAG AGA AAC ATG GTC ACA CCA GAA GGG 7794 Ala Lys Leu Arg Trp Phe Val Glu Arg Asn Met Val Thr Pro Glu Gly 2555 2560 2565
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE: ( ix ) FEATURE :
  • AAG GCG AAA AAC ACG CCT TTC AAT ATG CTG AAA CGC GAG AGA AAC CGC 162 Lys Ala Lys Asn Thr Pro Phe Asn Met Leu Lys Arg Glu Arg Asn Arg 10 15 20
  • CAG GGA CGA GGA CCA TTA AAA CTG TTC ATG GCC CTG GTG GCG TTC CTT 258 Gin Gly Arg Gly Pro Leu Lys Leu Phe Met Ala Leu Val Ala Phe Leu 40 45 50

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Abstract

L'invention concerne des clones d'ADNc infectieux servant à la constitution du virus de la dengue 2, souche 16681, ainsi que de son vaccin dérivé, atténué vivant, PDK-53 (DEN-2 PDK-53). L'invention concerne également des clones d'ADNc infectieux servant à la formation de virus chimériques caractérisés en tant que gènes structuraux d'expression d'un virus atténué de la dengue 1, dengue 3, ou dengue 4, dans le contexte des gènes non structuraux du virus de la dengue 2 PDK-53 (DEN-2/1, DEN-2/3, DEN-2/4). L'invention concerne en outre des produits de recombinaison génétiques codant ces ADNc, et des cellules hôtes contenant des produits de recombinaison. Sont également décrits des vaccins quadrivalents conférant l'immunité vis-à-vis des quatre sérotypes du virus de la dengue comprenant le dérivé des clones infectieux DEN-2 PDK-53, les virus DEN-2/1, DEN-2/1, DEN-2/3, ou DEN-2/4. L'invention concerne également des procédés d'immunisation associés.
PCT/US1996/009209 1995-06-07 1996-06-06 Virus pdk-53 infectieux de la dengue 2 utilise comme vaccin quadrivalent WO1996040933A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU60932/96A AU6093296A (en) 1995-06-07 1996-06-06 Infectious dengue 2 virus pdk-53 as quadravalent vaccine

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US48329295A 1995-06-07 1995-06-07
US08/483,292 1995-06-07

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Cited By (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999015692A2 (fr) * 1997-09-23 1999-04-01 Bavarian Nordic Research Institute A/S Antigenes du virus de la dengue et traitement de la dengue
WO1999034020A1 (fr) * 1997-12-31 1999-07-08 Akzo Nobel N.V. Dosage base sur la transcription isothermique permettant de detecter et de determiner le genotype du virus de dengue
WO2000057909A2 (fr) * 1999-03-26 2000-10-05 Walter Reed Army Institute Of Research Vaccin a base de virus de dengue-2 attenue
WO2000057907A2 (fr) * 1999-03-26 2000-10-05 Walter Reed Army Institute Of Research Vaccin multivalent contre le virus du dengue
EP1159968A1 (fr) * 2000-05-30 2001-12-05 Mahidol University Souches atténuées du virus de la Dengue et leur utilisation dans une composition vaccinale
EP1159969A1 (fr) * 2000-05-30 2001-12-05 Aventis Pasteur Vaccine composition
US6383488B1 (en) * 1997-01-15 2002-05-07 Centro De Ingeniera Genetic Y Biotechnologies (Cigb) Pre-M/M epitopes of dengue virus, synthetic peptides, chimeric proteins and their use
EP1263965A2 (fr) * 2000-02-16 2002-12-11 The Government of the United States of America, as represented by the Secretary, Department of Health & Human Services Flavivirus chimeriques avirulents et immunogenes
US7041255B2 (en) * 2001-03-01 2006-05-09 National Health Research Institute Detection of dengue virus
WO2006134443A1 (fr) * 2005-06-17 2006-12-21 Sanofi Pasteur Souche attenuee du serotype dengue 2
WO2008047023A2 (fr) 2006-10-04 2008-04-24 Sanofi Pasteur Methode d'immunisation contre les 4 serotypes de la dengue
US7641907B2 (en) 2005-06-17 2010-01-05 Sanofi Pasteur Dengue serotype 1 attenuated strain
EP2143440A1 (fr) 2008-07-09 2010-01-13 Sanofi Pasteur Agent stabilisant et composition vaccinale comprenant un ou plusieurs flavivirus vivants atténués
EP2353609A1 (fr) 2010-02-04 2011-08-10 Sanofi Pasteur Compositions et procédés d'immunisation
WO2014016362A1 (fr) 2012-07-24 2014-01-30 Sanofi Pasteur Compositions de vaccin pour prévenir une infection provoquée par le virus de la dengue
WO2014016360A1 (fr) 2012-07-24 2014-01-30 Sanofi Pasteur Compositions de vaccin
US8715689B2 (en) 2008-04-30 2014-05-06 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention Chimeric west nile/dengue viruses
WO2014083194A1 (fr) 2012-11-30 2014-06-05 Sanofi Pasteur Procédés d'induction d'anticorps
AU2012202659B2 (en) * 2000-02-16 2015-05-14 Mahidol University Avirulent, immunogenic flavivirus chimeras
US9295721B2 (en) 2008-03-05 2016-03-29 Sanofi Pasteur Sa Process for stabilizing an adjuvant containing vaccine composition
WO2016106107A2 (fr) 2014-12-22 2016-06-30 Merck Sharp & Dohme Corp. Compositions de vaccin contre le virus de la dengue et procédés pour les utiliser
WO2017109698A1 (fr) 2015-12-22 2017-06-29 Glaxosmithkline Biologicals Sa Formulation immunogène
AU2015213355B2 (en) * 2000-02-16 2017-10-05 Mahidol University Avirulent, immunogenic flavivirus chimeras
US9783579B2 (en) 2013-03-15 2017-10-10 Takeda Vaccines, Inc. Compositions and methods for dengue virus chimeric constructs in vaccines
US9861692B2 (en) 2013-06-21 2018-01-09 Merck Sharp & Dohme Corp. Dengue virus vaccine compositions and methods of use thereof
WO2019069130A1 (fr) 2017-10-05 2019-04-11 Sanofi Pasteur Compositions pour vaccination de rappel contre la dengue
CN111944104A (zh) * 2020-08-11 2020-11-17 华南师范大学 一种检测登革热ns1蛋白的多孔隙双模板分子印迹聚合物微球及其应用
US11426461B2 (en) 2018-09-05 2022-08-30 Takeda Vaccines, Inc. Methods for preventing dengue and hepatitis A
US11464815B2 (en) 2018-09-05 2022-10-11 Takeda Vaccines, Inc. Dengue vaccine unit dose and administration thereof
US11590221B2 (en) 2018-09-05 2023-02-28 Takeda Vaccines, Inc. Dengue vaccine unit dose and administration thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992003545A1 (fr) * 1990-08-15 1992-03-05 Virogenetics Corporation Vaccin a base de poxvirus recombine contre le flavivirus
WO1992003161A1 (fr) * 1990-08-27 1992-03-05 The United States Of America, Represented By The Secretary, United States Department Of Commerce Proteines d'enveloppe de flavivirus avec pouvoir immunogene accru utilisables dans l'immunisation contre les infections virales
WO1993006214A1 (fr) * 1991-09-19 1993-04-01 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Flavivirus chimeriques et/ou flavivirus a croissance limitee
WO1993022440A1 (fr) * 1992-04-29 1993-11-11 National University Of Singapore Sequence de l'adn complementaire du serotype 1 du virus de la dengue (souche singapour)

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992003545A1 (fr) * 1990-08-15 1992-03-05 Virogenetics Corporation Vaccin a base de poxvirus recombine contre le flavivirus
WO1992003161A1 (fr) * 1990-08-27 1992-03-05 The United States Of America, Represented By The Secretary, United States Department Of Commerce Proteines d'enveloppe de flavivirus avec pouvoir immunogene accru utilisables dans l'immunisation contre les infections virales
WO1993006214A1 (fr) * 1991-09-19 1993-04-01 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Flavivirus chimeriques et/ou flavivirus a croissance limitee
WO1993022440A1 (fr) * 1992-04-29 1993-11-11 National University Of Singapore Sequence de l'adn complementaire du serotype 1 du virus de la dengue (souche singapour)

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
41st Annual Meeting of the American Society of Tropical Medicine and Hygiene Washington, USA November 15-19 1992 *
BLOK, J. ET AL.: "Comparaison of Dengue -2 virus and its candidate vaccine derivative: sequence relationships with the Flaviviruses and other viruses", VIROLOGY, vol. 187, no. 4, April 1992 (1992-04-01), ORLANDO US, pages 573 - 590, XP000601641 *
GRUENBERG, A. ET AL.: "Partial nucleotide sequence and deduced amino acid sequence of the structural proteins of Dengue virus type 2, New Guinea C and PUO-218 strains", JOURNAL OF GENERAL VIROLOGY, vol. 69, no. 6, June 1988 (1988-06-01), pages 1391 - 1398, XP000600928 *
HAHN, Y.S. ET AL.: "Nucleotide sequence of Dengue 2 RNA and comparison of the encoded proteins with those of other flaviviruses", VIROLOGY, vol. 162, no. 1, January 1988 (1988-01-01), ORLANDO US, pages 167 - 180, XP000600931 *
RICO-HESSE, R.: "Molecular evolution and distribution of Dengue Viruses type 1 and 2 in nature", VIROLOGY, vol. 174, no. 2, February 1990 (1990-02-01), ORLANDO US, pages 479 - 493, XP002012813 *
VAUGHN, D.W. ET AL.: "Phase I testing of a dengue-2 live-attenuated vaccine strain 16681 PDK 53 in american volunteers", AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, vol. 47, no. 4 sup, 1992, pages 99 - 100, XP000600344 *
VAUGHN, D.W. ET AL.: "Testing of a Dengue 2 live-attenuated vaccine ( strain 16681 PDK 53) in ten american volunteers", VACCINE, vol. 14, no. 4, March 1996 (1996-03-01), GUILDFORD GB, pages 329 - 336, XP000579824 *

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WO1999015692A3 (fr) * 1997-09-23 1999-06-24 Bavarian Nordic Res Inst As Antigenes du virus de la dengue et traitement de la dengue
WO1999015692A2 (fr) * 1997-09-23 1999-04-01 Bavarian Nordic Research Institute A/S Antigenes du virus de la dengue et traitement de la dengue
AU754386B2 (en) * 1997-09-23 2002-11-14 Bavarian Nordic A/S Dengue virus antigens and treatment of dengue fever
WO1999034020A1 (fr) * 1997-12-31 1999-07-08 Akzo Nobel N.V. Dosage base sur la transcription isothermique permettant de detecter et de determiner le genotype du virus de dengue
US5968732A (en) * 1997-12-31 1999-10-19 Akzo Nobel, N.V. Isothermal transcription based assay for the detection and genotyping of dengue virus
US6333150B1 (en) 1997-12-31 2001-12-25 Akzo Nobel N.V. Isothermal transcription based assay for the detection and genotyping of dengue virus
WO2000057909A2 (fr) * 1999-03-26 2000-10-05 Walter Reed Army Institute Of Research Vaccin a base de virus de dengue-2 attenue
WO2000057907A2 (fr) * 1999-03-26 2000-10-05 Walter Reed Army Institute Of Research Vaccin multivalent contre le virus du dengue
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EP1263965A2 (fr) * 2000-02-16 2002-12-11 The Government of the United States of America, as represented by the Secretary, Department of Health & Human Services Flavivirus chimeriques avirulents et immunogenes
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EP1263965B1 (fr) * 2000-02-16 2011-09-28 The Government of the United States of America, as represented by the Secretary, Department of Health & Human Services Flavivirus chimeriques avirulents et immunogenes
US7094411B2 (en) * 2000-02-16 2006-08-22 The United States Of America As Represented By The Department Of Health And Human Services Avirulent, immunogenic flavivirus chimeras
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WO2016106107A2 (fr) 2014-12-22 2016-06-30 Merck Sharp & Dohme Corp. Compositions de vaccin contre le virus de la dengue et procédés pour les utiliser
US10449243B2 (en) 2014-12-22 2019-10-22 Merck Sharp & Dohme Corp. Dengue virus vaccine compositions and methods of use thereof
WO2017109698A1 (fr) 2015-12-22 2017-06-29 Glaxosmithkline Biologicals Sa Formulation immunogène
WO2019069130A1 (fr) 2017-10-05 2019-04-11 Sanofi Pasteur Compositions pour vaccination de rappel contre la dengue
US11426461B2 (en) 2018-09-05 2022-08-30 Takeda Vaccines, Inc. Methods for preventing dengue and hepatitis A
US11464815B2 (en) 2018-09-05 2022-10-11 Takeda Vaccines, Inc. Dengue vaccine unit dose and administration thereof
US11590221B2 (en) 2018-09-05 2023-02-28 Takeda Vaccines, Inc. Dengue vaccine unit dose and administration thereof
CN111944104B (zh) * 2020-08-11 2022-06-21 华南师范大学 一种检测登革热ns1蛋白的多孔隙双模板分子印迹聚合物微球及其应用
CN111944104A (zh) * 2020-08-11 2020-11-17 华南师范大学 一种检测登革热ns1蛋白的多孔隙双模板分子印迹聚合物微球及其应用

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