CA2420092A1 - New dengue and west nile viruses proteins and genes coding the foregoing, and their use in vaccinal, therapeutic and diagnostic applications - Google Patents

New dengue and west nile viruses proteins and genes coding the foregoing, and their use in vaccinal, therapeutic and diagnostic applications Download PDF

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CA2420092A1
CA2420092A1 CA002420092A CA2420092A CA2420092A1 CA 2420092 A1 CA2420092 A1 CA 2420092A1 CA 002420092 A CA002420092 A CA 002420092A CA 2420092 A CA2420092 A CA 2420092A CA 2420092 A1 CA2420092 A1 CA 2420092A1
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virus
recombinant
polypeptide according
west
sequence
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CA002420092A
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Philippe Despres
Frederic Tangy
Chantal Combredet
Marie Pascale Frenkiel
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Centre National de la Recherche Scientifique CNRS
Institut Pasteur de Lille
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Priority to CA002420092A priority Critical patent/CA2420092A1/en
Priority to CA002432738A priority patent/CA2432738A1/en
Priority to PCT/IB2004/001027 priority patent/WO2004076619A2/en
Priority to BRPI0407840A priority patent/BRPI0407840B8/en
Priority to ES04714865T priority patent/ES2394307T3/en
Priority to EP04714865.5A priority patent/EP1599495B9/en
Priority to CA002456873A priority patent/CA2456873A1/en
Priority to CA2517258A priority patent/CA2517258C/en
Priority to DK04714865.5T priority patent/DK1599495T3/en
Publication of CA2420092A1 publication Critical patent/CA2420092A1/en
Priority to IL170418A priority patent/IL170418A/en
Priority to US11/210,960 priority patent/US7556812B2/en
Priority to HK06105979.8A priority patent/HK1086278A1/en
Priority to US12/476,304 priority patent/US8859240B2/en
Abandoned legal-status Critical Current

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Description

NEW DENGUE AND WEST NILE VIRUSES PROTEINS ANp GENES C(~DINC~
ThIE IIrORE~DINta, ANG ThiEIR L.15E IN YACCINAL, 1'HERAPEIITIG
AND DIAfiNOSTIC APPLICATIONS
introduction The aim of this work is to develop viral vectors expressing different immunogens from the West Nite Encephalitis Virus (UVNV) or the Dengue virus able to induce protective humors! and cellular immune responses against LNNV or Dengue virus Infections. We describe 3 antigens from WNV (the secreted envelope glycoprotein (E}, the heterodimer glycoproteins {pre-Ivl-Ej and tile NS1 protein) and from Dengue virus (the secreted envelope glycoprotein (E), the heterodimer glycoproteins (pre~M-E) and the NS1 protein). These antigens have been derived from West Niie Virus 98-ST1 strain and from Dengue virus.
Any fragment of a r3ucleotidic or aminoacid sequence of the present invention comprising at least 30 nucleotides or 10 aminoacids are part of the present invention.
Any nucleotidic or aminoacid sequence having at least 80°~0 of identity of the sequences of the present invention are part of said inventian provided that the sequences have the capacity to hydridize under strir7gent conditions with the nucleatidic sequence as disclosed or the polynucieotidic sequences capable to be recognized by antibodies raised against any aminmacids sequences as disclosed in the present invention.
Various viral vectors are known and well documented. Ids exarrtpfes, we refer to the use of Hepatitis B virus (French Patent hI° 2636532), or Human Papilloma Virus, ar Polio virus (International patent application n°WO 89 D1516}, o~r Mengo virus (International patent application n°WG 94 2972), or Measles virus (European Patent Application N3 0229~5~1.6 flee on June 20, 2U~2} for the expression of foreign nucleotidic sequences as use#u1 immunogens in therapE:utical, vaccines, or diagnostic fields.
lr~troductic~n WNV
Ftaviviridae are arboviruses (arfhrvpc7d-borne virus} mainly transporketl by mosquitoes and blood-sucking ticks. They are small encapsid~ated viruses and their ~enomES consist of infectious singe-stranded anr! iine~r RNA of positive pt~larity. in y Y

Man, flaviviruses cause deadly Yremarrhagic fever or meningc~-encephalitis.
Yellow fever, dengue fever and Japanese encephalitis are the main, tropical flaviviroses.
ether' important human f9aviviroses are Saint houis encephalitis, tickrbarn European encephalitis and West Nile fever.
West Nile fever is a zoonosis associated with a flavivirus which was first isolated in Uganda in 1937. Its transmission cycle calls for birds as the main resevoir and for blood sucking mosquitoes of the Cut~x genus as vectors. Migratory virsmic birds transport the virus to far-away regions where they transmit ii: anew to ornithophile mosquitoes of the Culex genus. IVtany species of mammals ;are permissive for the West Niie virus. Horses are particularly sensitive t~ the disease but dcy riot participate irt the cycle of transmission. West Nile fever is endemic in Affica, Asia, Europe 'and Australia. Phylogenic studies have revealed the existence of fiwo strains of viruses viral line 1 has a worldwide distribution, and viral line ~ is essentially African. ~6iral line 1 was responsible far enzooties in Romania ~19g~), Russia (1999), Israel {1998-2000) and mare recently in Forth America where the virus had never been detected before 1999. The viral strains isolated during the recent epidemics in Israel and the United-States are more than 99,7 °/o identical. In the Diddle-East and Norkh America, where the virus has taken root, an important bird rrlortality rate has been observed among infected birds, notably in Caafviclae. In Nortlh America, over x#000 subjects were infected with the West Nile virus, ~5Q of which died between the months of August and December X002. At the present time, zoonosis is observed in all regions of the United States. At the moment, there exists no human vaccine or specific therapy against West Nile fever.
In temperate and subytropical regions, human infections may occur during the fall season. When a subject is bitten by an infected mosquitos, the incubation period larsts approximately one week but Iess than 20 °/a of people infected with the West Nile virus ever go on to clinical manifestations. In its benignant form, the viral infection manifests itself by an undifferentiated febrile state associated with muscular weakness, headaches and abdominal pain. In less than 1°!~, c~f infected subjects, encephalitis or acute aseptic meningitis may occur, Spl~enora~egaly, hepatitis, pancreatitis and myccarditis are also observed. Flask paralyses similar to a poliomyelitis syndrome have recently been reported, but fatal cases of viral encephalitis (b°l° of patients having severe neurological disorders) mainly concern fragile subjects and the aged. Inter-human transmission of tl~e virus has else recently been observed in the United-States in subjects having undergone organ transplants of having been perfused With canfaminated blood products. intro-uterine transmission of the virus has been reported in the United-States. The dev~elopmet~t.nf a hu.rn_a_n vaccine against the West Nile fever is a priority in view of the fact thi~t the zoonoSis has taken root in North America and is expected to propagate in the coming months to Central America, South America and the Caribbean where dengue fever and yelkaw fever are already rampanf.
The present invention will be more readily understood by referring tovthe i_Im...:~... _..._ -.1._.. T4.~~a ~.-...-te.. ..,. :11...-.t....a:.... t ih.... ~ ~ri.. ."-..s~f nr.nlin.ahifiW r of the present inventir~n and are not intended to limit its scope:
Itnodtttcattans ana variations ran be made theCein without deparking from the spirit and scope of the invention. Although any methods and materials similar or equivalent t4 those described herein can be used in the practice for testing of the present invention; the preferred methods and materials are described.
Example 1 : Construc~on of measles viruses (MV) expressing "WNV astd Den'I antigens In order to test their capacity as vaccine candidates against WNV infeotian;
we constructed recombinant Schwarz measles viruses (h~IV~ expressing these WNV:and DEN-1 antigens. The different genes were introduced in an additions( transcription unit in the Schwarz MV cDNA that we previously cloned ~pTl~~-MVSchw) (European Patent Application N° 022918a1.fi filed on June 20, 20U2). After rescue of the different recombin2~nt Slchwarz measles viruses expressing the WNV and DEN-1 genes, (heir capacity to protect mice from a lethal WNV intraperitoneal challenge,. and monkeys from Dengue virus infection will be tested.
MV vector Mass vaccination with live attenuated vaccines floe reducced the incidence of measles and its complications dramatically since it was intrc>'duced in the St)'s. By now, the vaccine has been given to billions of people and is safe and efFieaCious. It induces a very efficient, fife-long CI~~> CD8 and humoral immunity after a single injection of '! 04 T~ID~4. Moreover, it is easy to produce, cheap, and the means to deliver It worldwide already exist. TYte safety of this uaccine is clue to several factors:

!) The stability of the MV genome which explains that reversion to pathoget~icity has never been observed, ii)The impossibility for the MV genome to integrate in host chromosomes since viral replication is exclusively cytoplasmic. iii) The production of the vaccine on safe primary chick embryo fibrobtastic cefls_ Thus, live attenuated ;MV
could provide a safe and efficient pediatric vaccination vector.
MV belongs to the genus l~icrt;bidddvirr~s in the family f~aramyxoviridae. The ~dmonston MV was isolated in 1954 (1), serially passaged on primary human kidney and amnion cells, then adapted to chick embrys~ fibroblasts (CEF) to produce Edmonston A and B seeds (see (?, S) for review). Edmonston E was licensed in as the first MV vaccine. Further passages of ~dmonston ,~ and B on CEF
produced the more attenuated Schwarz and Moraten viruses (4) whose sequences trove recently been shown to be identical (5, 6). Being "reactogenic,'°
Edrnorrston S vaccine was abandoned in 1975 and replaced by the Schwar~IMoraten vaccine. This is now the most commonly used measles vaccine (2, 3).
In a previous work, we constructed an infectious cDNA frorrr a batch of commercial Schwarc vaccine, a widely used MV vaccine (European latent Application N° ~~29'1551_6 filed on June 20, ~~2). The extremit;es of the cDNA
were engineered in order to maximize virus yield during rescue. A previously described helper cell-based rescue system was adapted by co-cultivating tr'ansfected cells on primary chick embryo fibroblasts, the cells used to produce the Schwarz vaccine. After two passages the sequence of the rescued virus was identical to that Of the cDNA and of the published Schwarz sequence. Two additional transcription units {ATU) were introdurxd in She cDNA for cloning foreign genetic material.
The immunogenicity of rescued virus was studied in mice transg~;nic for the C1~4~6 MV
receptor and in macaques. Antibody titers in animals inoculated with low doses of the rescued virus were identical to those obtained with commercial Schwarz MV
vaccine.
In contrast, the immunogenicity of a previously described Edmonston strain-derived MV clone was much lower, This new molecular clone allows producing MV vaccine without having to rely on seed stocks. The ATtJs, allow producing recombinant vaccines based on an approved, efficient and worldwide used vaccine strain.
F~cample 2: Construction of ~chwarz M~'-VI~NV recombinant plasmids.
~) Secreted glyccrprateln E frr~r» YIdIVV

The WNV env gene encoding the secreted form of the protein was generated by RT-PCR amplification of viral RNA pucsfied from viral particles ('WNV IS-strain), The specific sequence was amplified wing Pfr~Turbo DNA polymerase ~Stratagerte} and specific primers that contain unique sites for suiasectuent cloning in pTM-~ItVSehw vector : HIV-VIINEnv~~ 5'-TATCGTACGATGAGAGTTCaTGTTTGTGGTGCTA--3' (BsiWt site underlined) and fUIV-WNEnv3 5'-ATAGCGCGCTTAGACAGCCTTGCCAACTGAr3' (BssNIJ site urtderiir~ed). A start and a stop colon were adtfed at both ends of the gene.
The Hrhole settuence generated is '138() nucleotides long, including the start and the stt~p colons and respects the rule of six", stipulating that the nuct~:otides number of MV
genome must be divisible by 6 (7, 8}. The Ertv protein thus generated contains its signal pcptido en N-term (18 aa) end no transmemE~r~np rp~inn Thm, If rPpr~:~Prsts. _.. _.._.. ... ..
amino acids 27~-T32 in WNV polyprotein and has the following sequence SEQIbN°1 ~
atgagagttgtgtttgtcgtgctattgcttttggtggccccagcttacagcttcaactgc;cttggaatgagcaacaga g acttattggaaggagtgtctggagcaacatgggtggatttggttctcgaaggcgacagctgcgtgactatcatgtct aaggacaagcctaccatcgatgtgaagatgwtgaatutg~aggdggtcaacctf~gcagaggxccgcar~ttg ctatttggctaccgtcagcgatctctcoaccaaagctgcgtgcccgaccatgggagaagctcacaatgacaaac gtgctgacccagcttttgtgtgcagacaaggagtggtggacaggggctggggcaacggctgaggattatttggca aaggaagcattgacacatgcgccaaatttgcctgctcta~aaggcaataggaa~gaaccatcttgaaagagaa JJ~J~c"actcaggcag9g~9~>~~'a~'g'c~"d~~~'~~t~~a~'c~'a'~Z~~~a~'~itC~d'g~d-"~''~~t~~ -agaggtgacagtggactgtgaaccacggtcagggattgacaccaatgcatactacgtgatgactgttggaacaa agacgttcttggtccatcgtgagtggttcatgg acctcaa cctcccttgg agcagtg ctg g aagtactgtgtg g egg aacagagagacgttaatggagtttgaggaaccacacgccacgaagcagtctgtgatagcattgggotcat;aag agggagctctgcatcaagctttggctggagccattcctgtggaattttcaagcaacactgtcaagttgacgtcgggt ~tttgaagtgtagagtgaagatggaaaaattgcagttgaagggaacaacctatc~gcgtctgttcaaaggctttGa agtttcttgggactcccgcagacac2tggtc~rcggcactgtggtgttggaattgcagtacactggcacggatggac cttgcaaagttcctatctcgtcagtggcttcattgaacgacctaacgccagtgggc<~gattggtcactgtcaaccctt t tgtttcagtggceacggcr~acgctaaggtcctgattgaattggaaccaccctttggagactcatacatagtggtgg gcagaggagaacaacagatcaatcacca#ggcacaagtctggaagcagcattggcaaagcctttacaacca ccctcaaaggagcgcagagactagccgctctaggagacacagct~ggacfttggatcagttggaggggtgitc acctcagttgggaaggctgtctaa SEt~ iD N° 5 i MRVVFVVLLLLVAPAYSFNCLGMSNF~DFLIGVSGATIWDLVI_EGt~SCVTIMSKD
KPTfDVKMMNMEAVhILAEVRSYCYI-ATVSDLSTKAACPTiIatGEAHNDKRADPAF
VGRQGWDRGWGNGCGLFGKGSIATCAKFACSTKAIGRTILKENlKYEVAIFVHG
PTT~/~SHGNYSTQVGATQAGRFS ITPAAPSYTLKL.GEYGEVI-VDC EPRSG I DTN
AYYVMTVGTKTFLVHREWFttrIDLNLPVVSSAGSTVWRNRETLMEFEEPHATKC~S
VIALGSQEGALHQALAGAIPVEFSSNTVKLTSGHLKCRVKMEKLC~LKGTTYGVC
SKAFKFLGTPApTGHGTWLELQYTGTaGPCK4lPISSVASLNDLTPVGRLV-fVl~
PFVSVATANAKVLIELEPPFGDSYIWGRGEQQINHHWHKSGSSIGKAFT'rTLKG
Af,~I~LAALGpTAWDFGSVGC~VFTSVGKAV'~
Z) preM plus E glycoprofeins from tNNV
The WNV gene encoding the preM plus E glycoprcteins was generated by PCR
ampl~cation of plasmid pVL prM-E.55.1 (clone CNCM I-273 depose le 15 Octobre ADO'!, Philippe Despres). This expression plasmid encodes the pre~M and E
proteins of WNV (6S-98-ST1 strain). The sequence was amplified using PfuTurbo DNA
polymerase (Stratagene} and specific primers that contain unique sites for subseguent cloning in pTM-MVSchw vector v MV-WNpreMES 5'-TATCGTACGATGGAAAAGAAAAGAGGAGGAAAG--3' (BsiWl site underlined) and MV-WNpreME3 5'.-ATAGCGGGGTTAAGCGTGCACGTTGACCaG AG-3' ~BssNll site underlined). A start and a step codan were added at both ends of the gene. The whole sequence generated is 2076 nuclet~tides long, ir~c9uding the start and the stop codons and respects the MV "rule of six". in this canstrcrct, the ~-terminus part of the C protein serves as a prM translocation signal. Bath preM ann E viral glycoproteins are transrnembrane giycoproteins type i. It is presumed ti~at WNV env preME
expressing MV will produce and release muitimeric forms of preM-E heterodimers exhibiting high immunagenic potential. The construct represents arrtino acids 3~D~-~$9 in WNV polyprotein and has the fDllowing sequence SEQ ID N° 2 ~ atgcaaaageaaagaggaggaaagaccggaattgcagtcatgattggcctga~tcgccagcgtaggagcagtt accctctctaacttccaagggaaggtgatgatgacggtaaatgctackgacgtcacagatgtcatcacgattccaa cagctgc~gaaagaacctatgcattgtcagagcaatggatgtgggatacatgtgcgatgatactatcaGttatga atgcccagtgctgtcggCtggtaatgatccagaagacatcgactgttggtgcaca~aagtcagcagtctacgtcag gtatggaagatgcaccaagacacgccactcaagacgcagtcgg aggtcactgacagtgc;agacacacggag aaagcactctagcgaacaagaagggggcttggatggacagcaccaaggccacaaggtatttggtaaaaaca gaatcatggatcttgaggaaccctggatatgccctggtggcagccgtcattggttggatgcttgggagcaacacc _.e:y,__"~_o_~u~,yLU.~ya.~,.ry..s~yy~Jlie.~~a~~ ~~~
=~i6......=...i.4...~.....,~..rr.niiw...r...&r.-mvnnnnra gagacttcttggaaggagtgtc~,qgagcaacatgggtggatttggttctcgaaggcgacagctgcgtgactatcat gtctaaggacaagcctaccatcgatgtgaagatgatgaatatggaggcggtcaa.cctggcagaggtccgcagtt attgctattfggctaccgtcagcgatctctccaccaaagctgcgtgcccgaccatgc~gagaagctcacaatgaca aacgtgctgacccagcttttgtgtgcagacaaggagtggtggacaggggctgg9gcaacggctgcggattatttg gcaaaggaagcattgacacatg Ggccaaatttgcctgctctaccaaggcaatag ga agaaccatcttgaaaga gaatatcaagtacgaagtggccatt~ttgtccatggaccaaCtactgtggagtcgc~acggaaactactccacaca ggttggagccactcaggcagggagattcagcatcactcctgcggcgccttcatacacactaaagctkggagaat atggagaggtg acagtggactgtgaaccacggtcagggattgacaccaatgcatactacgtgatg actgttg g a acaaagacgttcttggtccatcgtgagtggttcatggacctcaacctcccttggagcagtgctggaagtactgtgtg gaggaacagagagacgttaatggagtttgaggaaccacacgccacgaagcagtctgtgatagcattgggctca caagagggagctctgcatcaagcittggctggagccattcctgtggaattttcaagcaacactgtcaagttgacgtc gggtcatttgaagtgtagagtgaagatggaaaaattgCagttgaagggaacaac:ctatggcgtctgttcaaaggc tttcaagtttcttgggactcccgcagacacaggtcacggc~ctg~gtgttgg~attYr-.ant,~~:artnacacggata ._ _ . _ . . _..
gaccttgcaaagttcctatctcgtcagtggcttcattgaacgacctaacgccagtgc~gcagattggtcactgtcaac cctfttgtttcagtggccacggcc2~acgctaaggtcctgattgaattggaaccacccattggagactcatacatagtg gtgggcagaggagaacaacagatcaatcaccattggcacaagtctggaagcagcattggcaaagcctttaca accaccctcaaaggagcgcagagactagccgctctaggagacacagcttgggactttggatcagttggagggg tgttcacCtcagttgggaaggct9tccatcaagtgttcggaggagca~.ccgctcaa:.tgttcaggcatgtcctc~a ..
ataacgcaaggattgctgggggctctcctgttgtggatgggcatcaatgctcgtgataggtccatagctctcacgttt ctcgcagttggaggagttctgctcttcctCtocgtgaacgtgcacgcttaa SEQ !D N° 6 a M~KtCRGGKTGIAVMIGLIASVGAVTI-SNFQGKVMMTVNATDVTDV1TIPTAAGKN
ewr. r.nr."rwrwr~r.er.~o~~r~~rercwn~J'v.d4lL~Vr~~~l'~TllTl9lJ
SRRSRRSL'i'1iQ'1'HGESTL.AIVKKGAWMDSTKATRYLVKTESWILf~I~t-~c~YAwl~~t VIGWM~GSNTMQRVtIFVVLLLtVAPAYSFNCLGMSNRDFLEGV~GATWVDLVL
EGDSCVTlMSKDKPTIDVKMMNMI=AtINLA~VRSYCYLAT~JSDLSTKAACPTMGE
AHNDKRADPAFVCf~c~GVVDRG1NGNGCGLFGKGSIDTCAKFAC~TKAIGRTILI~

FNI~CYEVAIFVHC~P~ESF-~GNYSTQVGATC2~1GRFSlTPAAF'SYTLKLfaEYGEVT
vaCEPRSGIDTNAYYVMIV~TKTFLVHI~EwF~nDLNLPws,sAGS-rVWRNRETL
MEFEEPHATKQSVlALG~QEGALF~iQALAGA(PVEFSSNTVKLTSGHLKCRVKMI=
KLaLKGTTYGVCSKAFKFLGT'f'A~TGHGTVVLELQYTGTL?GPCKVPISGVA SLN
DLTPVGRLVTVNPFVGVATAi~AKVLI~LEPPFGDSYiVVGR.~EQQINHH1NHKSG
SS1G~<AFTTTLKGAQRLAALGDTAWDFGSVGGVFTSVGKAVHQV'FGGAFRSLF
GGMSWiTQGLL~ALLLVIINi~ftJARDRSfALTFL~VGGVLLFf_SVNVHA*
3~ N5? pmteirt from WNV
The WNV NS? gene was generated by RT~PCR amplification of viral RNA , purified from viral particles (WNV° 15-98~5T1 strain). The specific sequence was amplified using PfuTurbo DNA polymerase (~tratagene) and ;specific primers :
MV-WNNS15 5'- TATCGTACGATGAC3GTDGATAGCTGTCAt'>G-3° (BsiWI site underlined} and MV-WNh1S13 ATAGCGCGCTCATTAGGTCTTTTCATCATGTCT(:-3' (~sst~lJ site underlined}. A
start codan was added at the ~' end and two stop codons at the 3' end of the sequence. The whole sequence is 1110 nuCiet~tides long, including the start and the two step colons; thus respecting the "rule of six". The N~1 protein generated contains its signs! peptide sequence in N~terrn (~3 as}. It represents amino acids 769-1136 in WNV polyprotein and has the following sequence r~~~ ~~ N
atgaggtccatagGtctcacgtttctcgcagttggaggagttctgctcttcctctccgtgaacgtgcacgctgacactg ggtgtgccatagacatcagccggcaagagctgagatgtggaagtggagtgttcatacacaatgatgtggaggct tggatggaccggtacaagtattaccctgaaacgccacaaggcctagccaagatc;attcagaaagctcataagg aaggagtgtgcggtctacgatcagtttccagac#ggagcatcaaatgtgggaa9c;agtgaaggacgagctgaa eactcttttgaaggagaatggtgtggaccttagtgtcgtggttgagaaacaggagggaatgtacaagtcagcacc taaacgcctcaccgccaccacggaaaaattggaaattggctggaaggoctggg~gaaagagtattttatttgcac cagaactcgccaacaacacctt#gtggttgatggtccggagaccaac~gaatgtcc:gactcagaatcgcgcttgg aatagcttagaagtggaggattttggattkggtctcaccagcactcggatgttcctgaaggtcagagagagcaaca caactgaatgtgactcgaagatcattggaacggotgtcaagaacaacttggcgat:ccacagtgacctgtcctattg gattgaaagcaggctcaatgatacgtggaagcttgaaagggcagttctgggtgaagtcaa~ttcatgtacgtggc ctgagacgcataccttgtggggcgatggaa#ccttgagagtgacttgataataccagtcacactggcgggaccac gaagcaatcacaatcggagacctgggtacaagacacaaaaccagg9cccatc~ggacgaag9ccgggtag agatlgacttcgattactgcccaggaactacggtcaccctgagtgagagctgcggacaccgtggacctgccactc gcaccaccacagagagcggaaagttgataacagattggtgctgcaggagctgt:accttaccaccactgcgcta ccaaactgacagcggctgttggtatggtatggagatcagacoacagagacatg~~tgaaaagacctaatga SEA Ia P~° T
s MRSIALTFLAVGGVLLFLSVNVHADTGCAIDISRGtELRCtaL~GIfFIHNDVFAWNlDR
YKYYPETPQGLAK11QKAHKEGVG~3LRSVSRLEHQMWF~'uKDf=L.NTLLKENGVD
~.SWVEKQEGMYKSAPKRLTATTEKL.EICzWKAWGKSILF.~.PELANNTFVVDCaPE
TKEGPTQNRAWNSLEVEDFGFGLTSTRMF'LKVRESNTTECf~SKIIGTA~JKNNLAI
HSDLSYWIESRLNDTWKLERAVLGEVKSCTWPETp-ITLWGaDGILESDLIIPVTLAG
PRSNHNRRPGYKTQNQGPWI~FGR~tEIDFDYCP~TI'VTLSESC~~HRGPATRTT
TESGKLITDWCCRSCTLPPLRYC~TDSCaCVVYGMEIRPQRI-If~EKT""
4~ preM-E protein from Dengue type 9 virus The Dengue virus gene encoding the preM plus E glycoproteins was generated by PCR amplification of plasrt~id pVL pINaI~prM+E) (clone 2) I;CC)URAC3EC)T, M.-I~., FRENKIEL, M.-P., DUARTE DDS SANTC)S, C.N., DEUSEL., V. & DFSPRES P.
2000, A-glucosidase inhibitors reduce dengue virus production by affecting the initial steps of virion morphagenesis in the endoplasmic reticuium. ,;lournal of Virology ?'.4:
5~~-57~}. Tills plasmid encodes the pre-M and E glyccproteins of DEN-1 virus (strain FGAl89}. The sequence was amplified using PfuTur6o ~NA polymerase (Stratagene) arid specific primers that contain unique sites fr~r subsequent cloning in pTM-MVSchw vector : MV-pFN1 preMES 5'-TATC~TACGATGAACAGGAGGAAAAGATCCCTG-3' (BsiW! site underlined) and MV-DENIpreME3 5'-ATAGCGCGCTTAAAGCATGACTCCTACaGT ACAG-3' (Bss~'l!
site underlined}. A start and a stop colon were added at both ends of the gene. The whale sequence generated is X040 nucleotides long, including the start and the stop 4:ULlUffrl C&flCi-3t'.:rflt,.'-'G v=~~iv- 6r~;f='::a49-i,if=.3C:a:-'ii~-:~>~.-t:,'J.'.~'.Oe3'~',.t:SG~f=~.'~f-t.--~ ~~'..i~5#-?~.ix?-!'sF:r"~.--'~3~-~'".~~.
- __ C protein serves as a preM transDc~cation signal. Bath preM and ~ virral glycoproteins are transmemfJrane glycoproteiris type 1. It is presumed that f)EN-'! env expressing MV will produce and realease multirrteric farms of preM-E h~sterodirners exhibiting po~ypratem and nas the to~~owmg sequence 5~Q ID N° ~
atgaacaggaggaaaagatccgfigaccatgctcctcatgctgckgcccacagtcctggctttccatttgaccacac gagggggagagccacacatgatagttagtaagcaggaaagaggaaagtcactcttgttr"aagacctctgcagg tgtcaatatgtgcactctcattgcgatggatttgggagagttatgtgaggacacaat~gacktacaaatgcccccgga tcactgaggcggaaccagatgacgttgactgctggtgcaatgccacagacacai:gggtgacctatgggacgtgt tctcaaaccggtgaacaccgacgagacaaacgttccgtggcactggccccaca~cgtgggacttggtctagaaa caagaaccgaaacatggatgtcctctgaaggcgcctggaaacaaatacaaaaagtggagaCttgggctttgag acacccaggattcacggtgatagctcttttfttagcacatgccataggaacatccal:cactcagaaagggatcatttt catkctgctgatgctggtaacaccatcaatggccatgcgatgcgtgggaataggc~aacagagacttcgttgaagg actgtcaggagcaacgtgggtggacgtggtattggagcatggaagctgcgtcac:caccatggcaaaaaataaa ccaacatkggacattgaactcttgaagacggaggtcacgaaccctgccgtcttgcgcaaatkgtgcattgaagcta aaatatcaaacaccaccaccgattcaagatgtccaacacaaggagaggctacactggtggaagaacaagac gcgaactttgtgtgtcgacgaacggttgtggacagaggctggggcaatggctgcggactatttggaaaaggaag cctactgacgtgtgctaagttcaagtgtgtgacaaaactggaaggaaagatagttcaatatc~aaaaGttaaaatat tcagtgatagtcactgtccacacaggggaccagcaccaggtgggaaacgagactacagaacatggaacaatt gcaaccataacacctcaagctcctacgtcggaaatacagktgacagact2~cggaacccttacaotggactgctc acccagaacagggctg gactttaatgaggtggtgotattg acaatgaaagaaaaatcatgg cttgtccacaaac aatggtttctagactta~ccactgccttggacttcgggggcttcaacatcccaagagacttggaacagacaagatttg ctggtcacattcaagacagctcatgcaaagaagcaggaagtagtcgtactgggatcacaggaaggagcaatg cacactgcgttgaccggggcgacagaaatccagacgtcaggaacgacaacaatctttgcaggacacctgaaa tgcagattaaaaatggataaactgactttaaaagggatgtcatatgtgatgtgcacaggctcatttaagctagaga aggaagtggctgagacccagcatggaactgtcctagtgcaggttaaatacgaaggaacagatgcgccatgca agatccccttttcgacccaagatgagaaaggagtgacccagaatgggagattgataacagccaatcccatagtt actgacaaagaaaaaccaatcaacattgagacagaaccaccttttggtgagagctacatt:atagtaggggcag gtgaaaaagctttgaaactaagctggttcaagaaaggaagcagcatagggaaaatgttcgaagcaatcgccc 9a99a9cacgaaggatggctatcctgg9a9acaccgcatgggacttcggctctatagga~ggagtgtttacgtct gtgggaaaattggtacaccaggtttttggaaccgcatacggggtcctgttcagcgc,,~cgtttcttggaccatgaaaa t aggaatagggafcttgctgacatggttgggattaaatfcaaggagcgcgtcgcttt~cc~atgacgtgcatfgcagfit g gcatggttacactgtacctaggagtcatggtttaa "~ ') SEA ID 1~i 8 TLIAMDLGELCEDTMTYKCPRiTEAEP~DVDCWVCNATDTbWTYGTCSQTGEHR
RDKRSVALAPHVGLGLETRTETWMSSEGAWKt~IQKVETL'VALRHPGFTVIALFLA
HAIGTSITG~KGIIFILLIVILVTPSMAMRGVGIGNRDFVEGLSC9ATWVDVVLEHGSGV
TfMAKNKPTLI~IELLKTEVTNPAVI_RKLGIEAKiSNTTTDSR;~PTC~G~ATLVEEQD
ANFVCRRTVVDRGWGrJGCGLFrjKGSLLTGAKFKCVT'KLE:GK1V(~YENL.KYSVIV
TVHTGDC~HQVCaNETTEI-#GTIATITPC,ZAPTSEIQLTDYGTLI'LDCSI~RTGLDFNEV
VLLTMKEKSWLVHKc~WFLDLPLPWTSGASTSQETWNRQDLLVTFKTAHAKKQE
VVVLGSC~EGAMHTALTGATEIC~TSG I-TT9FAGHLKCRLKMDKLTLKGMSYVMCT
GSFKLEKEVAETc~.HGTVLVQVKYEGTDAPCKiPFSTC~DEKGVTQNGRLITANPIV
TDKEKPINIETEPPFGESYIIVGAGEKALKLSWFKKGSSIG~;~IIFEAIARGARRMAIL
GDTAWDFGSIGGVFTSVGKL\iHQVFGTAYtaVLFSGVBW TMKiG#GILLTWLGLN
SRSAS LSMTCIAVGMVTLYLGVMV*
The same immunogens can be pr~:pared by the same way from C1EN-2, DEN-~ .arid DE1~-~ serotypes.
5j Insertion into M1~ Schwarz vecfc~r The different WNV and ~EN-1 nuclec~tir~ic sequences were cloned ire pGR2.1-Tf~PO p9asmid {lnvitrogen~ and sequenced to cheok that no mutations were introduced. After BsillVlIBssHll digestion of the pCR2_1-TOPC7 plasmids, the fragments were cloned in the pTM-MVSchw ve~aor irr ATU position 2 giving plasrnids pTM-MVSchw-Env",,NV, pTM-MVSchw-preMEWn~, pTM-MVSchw-NSIwNV and pTM-MVSchW-preMEpEN-~ accordEng to Figure 1.

~,--.-..., ", a np ""~ 156D Gp y 2916 Cp CBJ-1 p"d!~

i.-..~T----- n 2asc by er~m _ &sN

r/ ~

~n;~'~ ~~ r--~.A =,i ~
1Da bA

~

j 7 hh (iE'I'U
pps 35 pTM-M'I~SChw- WNV

Figure 1. Schert~atic map of the pTM-MVSchw recambinant plasmids described ~xarn~le 3 . Recovery of recnr~binarrt ~lVSchvr-Ec~Vw,~v, MYSahw-~reMEw,"~ ~la~d MVSchw-NS9wNy viruses.
T~ reCOVef f~Ca,arTT~felalti .at;flYSiaTZ--~'3iu~rif vile ~~i~Si11id3;..~~-.UJy - ~~~-.. _I-- __....-._ helper~cell-based rescue system d2scribcd -by--Radec~ et-a'---{9;-~r~-r-:aodiffed b; ' -i Parks et al. {10}. Human helper cells stabiy expressing T7 RNA polymerase and measles N and P proteins (293-3-46 cells, a kind gift from IVIA Billeter) were ' transfected using the calcium phosphate procedure with pTM-tvlVSchw-!~nvurNV, pTM-M'1/''v-ChW-prBMEwnv or pTM-MVSchw-NSIu"~~ plasmids (5 pg~ a.nd a plasmid I
expressing the MV polymerase ~ gene {pEMC-La, ~0 ng, a kind gift from MA
Eilleter).
After overnight incubation at 37° C, the transfectian medium was replaced by fresh medium and a heat shock was applied (43° C for two hours) 1;10). Af~.er two days of Sr,.-~ ~~. .,+~.. .~+ QZ.° _ f fr f r~~,..g ) a rA tY C fr: f'I i ~ ~
C~?Ilfi. IFIVP..C ~11d i InGU~c7~eC~c9t JG Cr flC-uf~,s~! ~ cl~c~~~iy ~'uer~.rsti~wWi~illcW .I~o~a~~~'-~~wmsr msw.~.yw~u _ originally selected on CEF cells and is currently grown ,an these cells for safety considerations. Infectious ~rirus was easily recovered between 3 and ~ days following cocultivatiori. Syncytia appeared c~GCasianally in REF= but not systematically. The recombinant viruses were also resaaed by the same technique after cocultivation of transfected 293-3-~6 hetper cells at 3?° C with primate Vere~ cells {africart green monkey kidney. In this case, syncytia appeared systematic~~lly in call transfectiQns after 2 days of Goculture. In order to increase the yield crf rescue and because these recombinant viruses will be used in mice experiments, we usi::d Vert~ cells as target cells in place of the usual Ghick embryo fibrablasts (CE.F) (European Patent Application NG 022915~1.fi fifes on June 20, 2802). Recombinant virl~ses were i passaged two times on Vera cells. Mlle have previooasly shown that two passages ofi I
the Schwarz virus ors Vera cells did not change its immunogenic capacities in macaques (European Patent Application N° 022915b1.6 files on June ~0, 2002).
The recombinant viruses were prepared as described abcwe send the expression of the transgene in infected Gells was checked by immunofluc~rescence. To detect t WNV Envelope giycoproteins expression, we used irnrr~une sera from mice resistant to WNV infection (3nternatic~nal Patent Application Wc~ 0210817'4'1 ).'To detect NSs protein expression, we used anti-NS1 Monoclonal antiobadies {International Patent Application N° WO ~01756fi5).
>Biibhography WNV
Anderson, J.F., and Rahal, J.J. (2002). Efficacy of interferon alpha-?.b and ribavirin against West Nile virus infection.
Anonymous 0042)_ Intrauterine West Nile virus infection-New Ynric, 2002. MMINR
Morb Marta~ fNkly Rep. 51 :1135-11336.
Brintan, M.A. (2U02). The molecular biology of West tile virus: a nevv invader of the Western Hemisphere. Anna. Rev. NNcrabiat. 58: 371-x.42 Campbell, G., Marfin, A.A., Lanciotti, R.B., Cubler, 1~.J. {2002}. West Nile virus. Tfre Lancef 2: 519-529 I
Crupi RS, Asnis DS, Lee CC, Santucci T, Marino P~J, Flanz 5,.1. (2003).
Meet9ng the challenge of bioterrorism: Lessons learned from blest Nile virus and anthrax.
~4rn J
Emerg Med. 21:77-9.
Glass, J.D., Samuels, tJ,, Rich, M.M. (2002). Poliorr~yelitis due to Vllest Nile virus. N.
Engl. ,l. Med. Sep. 23 Lanciotti, R.S,, Ebei., G.D., Deubel, V., and al. {2002). Complete genome sequence analysis of West Nile virus strains isolated from the l.lnited States, ~:urope, anti the i Middie East. Virology 298 : 9B-105 Petsrsen, L.R., Raehrig, J.T., Hughes, J.M. {2p02). We5t NEIe vBCUS
encephalitis. dU_ I
Engl. J. Med. 347: 1225-1226 I
G~uirk, M. (2002). First treatment trio! for West Nils infection faegins.
f'lre f.anCet 2 : j 589 i i Tesh RB, Arroyo ,t, Travassas Da Rosa A~, Guzman H, Xiao SY, Monath TP.
(2002). , Efficacy of kiiled vircs vaccine, live attenuated chimeria virus vaaeine, and passive i immunization far prevention of West Nile virus encephalitis in hEamster rnadel. ~ri~erg lrtf~ct Dis. $:1392-1397.

~n~th TP, Arroyo J, Miller C, Guirakhoo F. {2001). West Mile virus vaccine.
burr ag Targets Infect ~isord. 1 :37-50.
~Iingraphy ~tIV
I=nders, .~. F. & Peebles, T. ~. (1954) Proc. hoc. Exp. Bic~d. fed. 86, 277-286.
griffin, D. (2c701} in Field's V!t'o~ogy, 4th ~difaon, eds. Knipe, fib. &
Howiey, P.
(i-ippincott - Raven Publishers, Philadelphia)" Voi. 2, pp. 14Q1-1 X4'1.
Hiifeman, M. (2002} V~ccirre 20, &51-665.
Schwarz, A. {1962) Arn. .i. Des. ~h~ld. 103, 216-2'99.
Parks, G. L., Lerch, R. A., V'dalpita, P., Wang, I~i. P., Sidhu, . S. 8 Udern, S_ (2061 ) J Vrrol 75, 910-20.
Parks, C. L., Lercn, F~. A., V~dalpita, P., Wang, t-!. P., Sid~hu, M. S. &
Udem, S.
(26Q1} J VfreJTS, 921-33.
Caiain, P. ~ Raux, L. (1993) J ldirc~! 67, 482-30.
Sehnei~let', H., Kaeiin, K. ~ Billeter, iVl. A. {1997) 'Jirology~ X27, 3.14-22.
Radecke, fF., Spielhof~r, P., Schneider, H., Kaelin, K., fiut~er, M., Dotsch, C., Christiansen, G. & Biileter, M. A. {1995) Emb~ J 14, 577;3-84.
Perks, C. ~., Lerch, R. A.. llltaipita, P., Sidhc~, M. ~. & Udem, S. A. (1999) J
of 73, 3560-C.

Claims (26)

1. A purified polypeptide wherein it derives from a West-Nile virus antigen or a Dengue virus antigen.
2. The polypeptide according to claim 1, wherein it is capable of inducing a protective immune response against a West-Nile virus or a Dengue virus in a mammal.
3. The polypeptide according to claim 1 or 2, wherein the West-Nile virus antigen is selected from the group consisting of secreted envelope glycoprotein (E), heterodimer glycoproteins (PreM-E) and NS1 protein.
4. The polypeptide according to claim 3, wherein the secreted envelope glycoprotein (E) comprises the sequence of SEQ ID NO: 5 or a fragment
5. The polypeptide according to claim 3, wherein the heterodimer glycoproteins (PreM-E) comprises the sequence of SEQ ID NO: 6 or a fragment thereof.
6. The polypeptide according to claim 3, wherein the NS1 protein comprises the sequence of SEQ ID NO: 7 or a fragment thereof.
7. The polypeptide according to claim 1 or 2, wherein the Dengue virus antigen is selected from the group consisting of secreted envelope glycoprotein (E), heterodimer glycoproteins (PreM-E) and NS1 protein.
8. The polypeptide according to claim 7, wherein the heterodimer glycoproteins (PreM-E) comprises the sequence of SEQ ID NO: 8 or functional derivatives thereof.
9. The polypeptide according to any one of claims 1 to 8, which is an immunogenic peptide.
10. A purified polyclonal or monoclonal antibody capable of specifically binding to a polypeptide according to any one of claims 1 to 9, or to a fragment thereof.
11. An expression vector comprising a polynucleotide sequence coding for a polypeptide according to any one of claims 1 to 9.
12. A purified polynucleotide sequence coding for a polypeptide according to any one of claims 1 to 9.
13. The purified polynucleotide sequence of claim 12 comprising a sequence selected from the group consisting of SEQ ID Nos: 1 to 4.
14. Use of a polynucleotide sequence as defined in claim 12 or 13 for detecting the presence or absence of a West-Nile virus antigen or a Dengue virus antigen in a biological sample.
15. A recombinant viral vector which is a recombinant virus comprising a polynucleotide sequence as defined in claim 12 or 13.
16. The recombinant viral vector of claim 15, wherein the recombinant virus is a live attenuated virus or a defective virus.
17. The recombinant viral vector of claim 15 or 16, wherein the recombinant virus is selected from the group consisting of measles virus, hepatitis B virus, human papillomavirus, picornaviridae and lentivirus.
18. A recombinant measles virus capable of expressing a polypeptide according to any one of claim 1 to 9.
19. A recombinant measles virus comprising, in its genome, a polynucleotide according to claim 12 or 13.
20. The recombinant measles virus of claim 18 or 19, which is a live attenuated virus or a defective virus.
21. A pharmaceutical composition comprising:
a) at least one component selected from the group consisting of:
a polypeptide according to any one of claims 1 to 9 or a fragment thereof;
- an antibody according to claim 10;
- an expression vector according to claim 11;
- a polynucleotide according to claim 12 or 13, - a recombinant viral vector according to any cane of claims 15 to 17;
and - a recombinant measles virus according to any one of claims 18 to 20;
and b) a pharmaceutically acceptable vehicle or carrier.
22. The pharmaceutical composition of claim 21, capable of inducing a protective immunity against a West-Nile virus or a Dengue virus in a mammal or in a bird.
23. Use of a pharmaceutical composition according to claim 21, as an anti-West-Nile virus agent, or for the preparation of an anti-West-Nile virus vaccine.
24. Use of a pharmaceutical composition according to claim 21, as an anti-Dengue virus agent, or for the preparation of an anti-Dengue virus vaccine.
25. A host cell incorporating an expression vector as defined in claim 11 or a recombinant viral vector as defined in any one of claims 15 to 17.
26. Method of producing a recombinant virus for the preparation of an anti-West-Nile virus vaccine or an anti-Dengue virus vaccine, the method comprising the steps of:
a) providing a host cell as defined in claim 25;
b) placing the host cell from step a) in conditions permitting the replication of a recombinant virus capable of expressing a polypeptide according to any one of claims 1 to 9; and c) isolating the recombinant virus produced in step b).
CA002420092A 2003-02-26 2003-02-26 New dengue and west nile viruses proteins and genes coding the foregoing, and their use in vaccinal, therapeutic and diagnostic applications Abandoned CA2420092A1 (en)

Priority Applications (13)

Application Number Priority Date Filing Date Title
CA002420092A CA2420092A1 (en) 2003-02-26 2003-02-26 New dengue and west nile viruses proteins and genes coding the foregoing, and their use in vaccinal, therapeutic and diagnostic applications
CA002432738A CA2432738A1 (en) 2003-02-26 2003-06-20 New dengue and west nile viruses proteins and genes coding the foregoing, and their use in vaccinal, therapeutic and diagnostic applications
CA2517258A CA2517258C (en) 2003-02-26 2004-02-26 Recombinant measles virus comprising dengue or west nile virus polynucleotides, and their use in vaccinal and therapeutic applications
BRPI0407840A BRPI0407840B8 (en) 2003-02-26 2004-02-26 recombinant measles virus, pharmaceutical composition, use of a pharmaceutical composition, bacterial cell, method for producing a recombinant virus and use
ES04714865T ES2394307T3 (en) 2003-02-26 2004-02-26 Proteins and genes of the West Nile virus and dengue and its therapeutic application
EP04714865.5A EP1599495B9 (en) 2003-02-26 2004-02-26 Dengue and west nile virus proteins and genes and their therapeutic application
CA002456873A CA2456873A1 (en) 2003-02-26 2004-02-26 West nile virus proteins and genes coding the foregoing and their use in vaccinal, therapeutic and diagnostic applications
PCT/IB2004/001027 WO2004076619A2 (en) 2003-02-26 2004-02-26 New dengue and west nile viruses proteins and genes coding the foregoing, and their use in vaccinal, therapeutic and diagnostic applications
DK04714865.5T DK1599495T3 (en) 2003-02-26 2004-02-26 PROTEINS AND GENES OF DENGUE AND WEST NILE VIRUS AND THERAPEUTIC APPLICATION THEREOF
IL170418A IL170418A (en) 2003-02-26 2005-08-22 Recombinant virus which is a live attenuated or a defective measles virus, pharmaceutical composition comprising same, its use and method of preparation
US11/210,960 US7556812B2 (en) 2003-02-26 2005-08-25 Dengue and West Nile viruses proteins and genes coding the foregoing, and their use in vaccinal, therapeutic and diagnostic applications
HK06105979.8A HK1086278A1 (en) 2003-02-26 2006-05-24 Dengue and west nile virus proteins and genes and their therapeutic application
US12/476,304 US8859240B2 (en) 2003-02-26 2009-06-02 Dengue and West Nile viruses proteins and genes coding the foregoing, and their use in vaccinal, therapeutic and diagnostic applications

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8475808B2 (en) 2003-05-23 2013-07-02 Novartis Vaccines And Diagnostics, Inc. Immunogenic reagents from west nile virus

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8475808B2 (en) 2003-05-23 2013-07-02 Novartis Vaccines And Diagnostics, Inc. Immunogenic reagents from west nile virus

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