CA2327189A1 - Novel dna-based vaccine against the encephalitis alphaviruses - Google Patents

Abstract

This invention relates to the development of a mammalian expression vector, under which expression of the structural genes of western equine encephalitis virus have been placed under the control of an eucaryotic promoter. When the recombinant vector is administered to mammalian cell culture or using a cell-free transcription/translation system, in vitro, authentic structural proteins of western equine encephalitis virus are produced as verified by reactivity with monoclonal antibodies developed to western equine encephalitis virus. When the recombinant DNA molecule is administered in vivo, a protective immune response is induced, thereby enhancing protection of the individual against subsequent infection by western equine encephalitis virus. In a similar manner, DNA vaccines to related alphaviruses (Venezuelan and eastern equine encephalitis viruses) could also be developed.

Description

Novel DNA-based Vaccine Against the Encephalitis Alphaviruses Field of the Invention This invention relates to the cloning, sequencing and expression of the structural genes of western equine encephalitis (WEE) virus strain 71 V-1658 and the development and use of the DNA-based vaccine against WEE.
Background of the Invention List of Prior Art Literatures Ausubel, F.M., et al, editors. (1995). C.'urrent Protocols in Molecular Biology, New York: John Wiley & Sons.
Bell, J. R., Bond, M.W., Nukapiller, M. B., Strauss, E.G., Strauss, J. H., Yamamoto, K., &
Simizu, B. (1983). Structural proteins of western equine encephalitis virus:
amino acid compositions and N-terminal sequences. Journal of !'irology 45, 708-714.
Bird, B.R. & Forrester, F.T. (1981 ). Basic Laboratory Techniques In Cell Culture. Atlanta:
U. S. Department of Health and Human Services, Centers for Disease Control.
Calisher, C.H. & Karabatsos, N. (1988). Arbovinus serogroups: definition and geographic distribution. In The Arboviruses: Epidemiologs acrd 1'cology, Vol. I,.pp. 1 '7-57. Edited by T. P
Monath. CRC Press: Boca Raton, Fl.
Calisher, C. H., Shope, R.E, Brandt, W., Casals, J., Karabatsos, N., Murphy, F.A., Tesh, R.B., & Wiebe"M.E.. (1980). Proposed antigenic classification of registered arbovirusess.
Intervirology 14, 229-232.
Calisher, C. H., Karabatsos, N., Lazuick, J. S.. Monath, T.P., & Wolff, K.L.
(1988).

Reevaluation of the western equine encephalitis antigenic complex of alphaviruses (family Togaviridae) as determined by neutralization tests. American Journal of Tropical Medicine and Hygiene 38, 447-452.
S Cilnis, M.J., Kang, W. & Weaver, S.C. (1996). Genetic conservation of Highlands J viruses.
Virology 218, 343-351.
Frohman, M.A., Dush, M.K. & Martin, G.R. (1988). Rapid production of full-length cDNAs from rare transcripts: Amplification using a single gene-specific oligonucleotide primer.
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Hahn, C. S., Lustig, S., Strauss, E.G. & Strauss, J.H. (1988). Western Equine Encephalitis virus is a recombinant virus. ProcE~edings of the National Academy of Science USA 85, 5997-6001.
Johnson, R.E. & Peters, C.J. (1996). Alphaviruses. In Fields Virology, 3rd edn, pp. 843-898.
Edited by B. N. Fields, et al., New York: Raven Press.
Kuhn, R., Hong, Z. & Strauss, J.H. (1990). 11!Iutagenesis of the 3' nontranslated region of Sindbis virus RNA. Journal of Virology 64, 1465-1476.
Kuhn, R.J., Niesters, H.G.M., Hong, Z. & Strauss, J.H. (1991). Infectious RNA
transcripts from Ross River virus cDNA clones and the construction and characterization of defined chimeras with Sindbis. Virology 182, 430-441.
Krieg, A.M., Yi, A.-K., Schorr, J. and Davis, H.L. (1998). The role of CpG
dinucleotides in DNA vaccines. Trends Microbial. 6, 23-27.
MeCluskie, M.J., Davies, H.L. (1999). Novel strategies using DNA for the induction of mucosal immunity. Critic. Rev. in Imrnunol. 19, 303-329.

Ou, J.-H., Trent, D.W. & Strauss, J.H. (1982). The 3' non-coding regions of alphavirus RNAs contain repeating sequences. Journal of Molecular Biology 156, 719-730.
0u, J-H., Strauss, E. G. & Strauss, J.H. (1983). The 5' terminal sequences of the genomic RNAs of several alphaviruses. Journal of Molecular Biology 168, 1-15.
Pardon, DR, Beckering, AM. (1997). Exposing the immunology of naked DNA
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Pfeffer, M., Proebster, B., Kinney, R.M. & Kaaden, O-R. (1997). Genus-specific detection of alphaviruses by a semi-nested reverse transcription reaction. American Journal of tropical Medicine and Hygiene 57, 709-718.
Pfeffer, M., Kinney, R.M. & Kaaden, O-R. (1998). The alphavirus 3'-nontranslated region:
Size heterogeneity and arrangement of repeated sequence elements. Virology 240, 100-108.
Prayaga, S.K., Fuller, D.H., Haynes, J.R. & Murphey-Corb, M. (1995). Particle-mediated nucleic acid immunization. Vaccines' 95, 105-109.
Reisen, W.K. & Monath, T.P. (1988). Western equine encephalomyelitis, pp. 89-137. In The Arboviruses: Epidemiology and Ecology, Vol. v. Edited by T.P. Monath. CRC
Press: Boca Raton, Fl.
Robinson, H.L., Feltquate, D.M., Morin, M.J., Haynes, J.R., Webster, R.G.
(1995). DNA
vaccines: A new approach to immunization. l/accine 95:69-75 Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular Cloning, a Laboratory Manual, ?nd edn.. Cold Spring 1-larbor: Cold Spring Harbor Laboratory.
Schlesinger, S. & Schlesinger, M.J. (1996). Togaviridae: The viruses and their replication, In Fields Virology, 3rd edn, pp. 825-84l . Edited by B. N. Fields, et al.. New York: Raven Press.
_;_ Strauss, J. H., & Strauss, E.G. (1988). Evolution of RNA viruses. Annual Review of Microbiology 42, 657-683.
Strauss, J. H., & Strauss, E.G. (1994). The alphaviruses: gene expression, replication, and evolution. Microbiological Review 58, 491-562.
Strauss, E.G., Rice, C.M. & Strauss, J.H. (1983). Sequence coding for the alphavirus nonstructural proteins is interruptec:l by an opal termination codon.
Proceedings of the National AcademyofScience USA 80, 5271-5275.
Strauss, E.G., Rice, C.M. & Strauss, J.H. (1984). Complete nucleotide sequence of the genomic RNA of Sindbis virus. Virology 133, 92-110.
Trent, D.W., & Grant, J.A. (1980). A comparison of new world alphaviruses in the western equine encephalomyelitis complex by immunochemical and oligonucleotide fingerprint techniques. Journal or Geraeral Virology 47:261-282.
Weaver, S. C., Hagenbaugh, A., Bellew, L.A., Netesov, S. V. , Volchokov, V. I.
, Chang, G.-J
J., Clarke, D. K., Gousset, L., Scott, T. W., 'Trent, D. W. & Holland, J. J.
(1993). A
comparison of the nucleotide seqeunces of eastern and western equine encephalomyelitis viruses with those of other alphaviruses and related RNA viruses. Virology 197, 375-390.
Weaver, S. C., Kang, W, Shirako, Y., Rumenapf, T., Strauss, E.G. & Strauss, J.H. (1997) Recombinational history and molecular evolution ofwestern equine encephalomyelitis complex alphaviruses. Journal of Y~rolo~- 71, 613-623.
Wolff, J.A., Malone, R.W., Williams, P., Chong, W., Acsasi, G., Jani, A., Felgner, P.L.
(1990). Direct gene transfer into mouse muscle in vivo. Science 247,1465-1468.
The alphaviruses are a group of about 27 enveloped viruses with a positive sense, nonsegmented single-stranded RNA genome (Calisher et al., 1980; Strauss and Strauss, 1988).
The alphavirus disclosed in this invention, western equine encephalitis virus (WEE), is amember of the WEE antigenic complex and is serologically related to the Sindbis (SIN), Highlands J (HJ), Fort Morgan, Buggy Creek, and Aura viruses (Calisher & Karabatsos, 1988;
Calisher et al., 1988). WEE is endemic in western North America and strains/varieties have been isolated from Argentina (AG80-646), Brazil (BeAr 102091 ) and the former Soviet Union (Y62-33) (Johnson and Peters, 1996; Weaver et al., 1997). In nature, WEE is transmitted from its amplifying hosts or reservoir in wild birds, to man and horses, by mosquitoes (Culex tarsalis being the principal vector). While the endemic cycle has resulted in only a limited number of human infections in recent years, in the past, major epidemics of WEE have been recorded. The most extensive epidemic, including 3,336 recognized human cases and 300,000 cases of encephalitis in horses and mules, occurred in the western United States and Canada in 1941 (Reisen &
Monath, 1988;
Johnson and Peters, 1996).
All alphaviruses share a number of structural, sequence, and functional similarities, including a genome with two polyprotein gene clusters (reviewed in Strauss &
Strauss, 1994;
Schlesinger & Schlesingerl 996). The genomic organization of these viruses is conserved (see Figure 1 ), with the nonstructural proteins translated directly from the 5' two-thirds of the genomic RNA. A subgenomic positive-stranded RNA (the 26S RNA), is identical to the 3' one-third of the genomic RNA and serves as the translational template for the structural proteins (capsid, E3, E2, 6K and E 1 ).
The nonstructural proteins (nsPl, nsP2, nsP3 and nsP4) are also synthesized as a polyprotein and processed into the four nsPs by a nsP2 protease. Two versions of the nonstructural polyprotein are synthesized in alphavirus-infected cells, due to frequent readthrough of an opal codon between the nsP3 and nsP4 genes in several alphaviruses (Strauss _5_ et al., 1983). The nsPs function in a complex with host factors to replicate the genome and transcribe the subgenomic mRNA. Alphaviruses have characteristic conserved sequences at the extreme S' and 3' domains and the intergenic region (0u et al., 1982, 1983;
Pfeffer et al., 1998).
These conserved domains are required for viral growth and replication and are believed to be important in promotion of protein synthesis and the initiation of RNA-dependent RNA
polymerise activity.
The relationship of different WEE isolates to each other has been demonstrated using neutralization tests (Calisher et al., 1988). Additionally, several strains of WEE were typed by oligonucleotide fingerprinting, and found to have greater than 90 % nt homology (Trent & Grant, 1980). The N-terminal sequences of the nucleocapsid, and the E1 and E2 glycoproteins have been determined by Edman degradation, and the E 1 and E2 proteins were found to have 82 and 71 % homology, respectively, to SIN (Bell et al., 1983). Hahn et al.
(1988) sequenced the 26S region of WEE strain BFS 1703. They proposed that WEE originated as a hybrid virus, formed by recombination of an EEE and a Sindbis-like virus, most likely during a co-infection event. They suggested that two crossover events occurred, one within the E3 gene, the other within the 3' nontranslated terminal region (NTR), resulting in a virus whose nonstructural domain, intragenic region, and capsid protein are similar to EEE, with envelope proteins showing homology to SIN.
Weaver et al. ( 1993 ) sequenced part of the nonstructural domain (nsP2 and nsP3 genes) of strain 5614, demonstrating this area also shows homology to EEE. Short regions within the nsP4 gene and the E1 protein/3' N'fR have been determined for many WEE
strains, allowing a preliminary assessment of the nucleic acid phylogenetic relationships within the WEE antigenic complex (Weaver et al., 1997). Serological studies (C'alisher et al., 1988) and preliminary sequence determination (Cilnis et cal., 1996; Weaver et al., 1997) ofthe HJ
genome suggests this is another closely related virus, and most likely a descendant of the same recombinant viral ancestor as modern WEE.
A highly conserved region of the alphavirus nsPl gene has been identified, and proved suitable for use in a polymerase chain reaction (PCR)-based genetic assay for alphaviruses, including WEE (Pfeffer et al., 1997). Phylogenetic analysis of this PCR
fragment yielded similar results to those obtained by Weaver et al., (1997) for a PCR fragment in the nsP4 gene.
In terms of therapy or prophylaxis, there are very limited possibilities. An inactivated vaccine to WEE is under investigational new dmg (IND) status. The vaccine uses formalin-inactivation of cell culture supernatants from WEE-infected tissue culture. It requires a minimum of 3 doses, yearly monitoring of antibody titer and possible boosters.
Its effectiveness in the protection against an aerosol challenge of WEE has yet to be established. A WEE live attenuated vaccine based on an infectious clone is under development (J.
Smith, personnel communication). The area of DNA immunization is relatively new, and has been reviewed in Hassett and Whitton, 1996; Donnelly et cil, 1997. Similar to live, attenuated vaccines, DNA
vaccines are known to stimulate both humoral and cellular immune responses (Pardon and Backering, 1997; McCuskie and I)avies, 1999). Much of the focus has been on methods to deliver and efficiently express the cloned products. lntramuscular administration of DNA has been one of the original methods used (Wolff et al, 1990). A second method uses ballistic delivery of DNA coated gold panic les, using high pressure helium gas to propel the particles into the epidermis and dermis of animals (Prayaga et al, 1995, reviewed by Robinson et al, 1995).
The Applicant identified a number of related areas of research, including the development of subunit vaccines to WEE. In the present invention, the Applicant disclosed the cloning, sequencing and expression of the stnrctural genes of a WEE virus (strain 71 V-1658), as described in Netolitzky et al., (2000) "Complete genomic RhIA sequence of western equine encephalitis virus and expression of the structural genes." ,lournc~l of Gejaeral virolo~,ry 81, 151-159. The DNA construct (pCXH-3), and a second construct (pVHX-6j were used in DNA
immunization studies in a mouse model for protection against intranasal administered WEE.
Summary of the Invention The present invention is directed to the development of a DNA-subunit vaccine to the WEE virus and its use against such virus. More specifically, DNA to structural components of the WEE virus are expressed and used as the subunit vaccine.
The present invention provides for the complete nucleotide sequence of WEE
strain 71 V-1658. Two novel cDNA clones, pCXH-3 and pVHX-6 are also disclosed as effective vectors for gene expression.
The present invention also provides the complete nucleotide sequence for the structural gene pcDWXH-7.
It further provides for a process for preparing a recombinant DNA vaccine against 1 S WEE virus, comprising cloning and sequencing of 265 region of a WEE virus strain 71 V-1658 under conditions suitable to effect in vitro transcription and translation of the functional recombinant DNA expression vector pCXH-3 and pVHX-6.
Brief Description of the Drawings Figure 1~ Diagram showing the WEE 71V-1658 sequencing strategy. 'The location of PCR
probe sequences used to screen the; WEE cDNA library are also indicated, along with the genomic organization of the virus.
Figure 2. Multiple sequence alignment.
_g_ Figure 3. Stem loop structures in thc~ S' NTR.
Figure 4. Stem loop structures in the 3' NTR.
Figure 5. Phylogenetic relationship of the WEE nonstructural region compared to other alphaviruses.
Figure 6. Expression of WEE stmctural genes in cell culture.
Figure 7. In vitro transcription and translation of WEE expression vectors.
Figure 8. WEE mouse infectivity model.
Figure 9. Protection using ballistic delivery of pCXH-3.
Figure 10. Protection using ballistic delivery of pVl IX-6 Figure 11. Protection using ballistic delivery of pVHX-6.
Detailed Description of the Invention The complete nucleotide sequence of tile 71 V-1658 strain of western equine encephalitis (WEE) virus was determined (minus vtwenty-five nucleotides from the 5' end) and shown in SEQ
ID NO: 1. A 5' RACE reaction was used to sequence the 5' terminus from WEE
strain CBA87.
The deduced WEE genome was 11,5()8 nucleotides in length, excluding the S' cap nucleotide and 3' poly(A) tail. The nucleotide composition was 28 ~~ A, 25 % C, 2S % G and 22 % U residues.
Comparison with partial WEE sequences of strain 5614 (nsP2-nsP3 of the nonstructural region) and strain BFS 1703 (26S structural region) revealed comparatively little variation; a total of 149 nucleotide differences in 8624 bases ( 1.7 °/. divergence), of which only 28% of these changes (42 nucleotides) altered the encoded anuino acids. Comparison ofdeduced nsPl and nsP4 amino acid sequences from WEE with the con-esponding proteins from eastern equine encephalitis (EEE) yielded identities of 84.9 % and 83.8 %, respectively. Previously uncharacterized stem loop structures were identified in the nontranslated terminal regions.
A 3100 by clone was identified (pcDNA-12) from the 3' end of the structural genes. A
1500 by fragment was PCR amplified and cloned into t:he 5' end of pcDNA-12 to produce a complete clone of the structural genes (XH-7) as shown in SEQ ID NO: 2. A cDNA
clone (pCXH-3) in which the structural genes of WEE strain 71 V-1658 were placed under the control of a cytomegalovirus promoter was made, and transfected into tissue culture cells. The viral envelope proteins were functionally expressed in tissue culture, as determined by histochemical staining with monoclonal antibodies which recognize WEE antigens. The construct was used to immunize mice ballistically and intramuscularly. Mice protected ballistically had a significantly reduced risk of infection, against a subsequent intranasal challenge with WEE
virus. A new vector was constructed to determine if increased levels of expression could be obtained. The construct used a pVAX vector to express the WEE structural genes (pVHX-6).
Upstream portion of the pVHX-6 vector to where it becomes the XH-7 sequence is shown as SEQ ID
NO: 3. The remaining nucleotide sequence of pVHX-6 from the point of divergence is identical to that of structural gene pcDWXH-7 of SEQ 1L) NO: 2.
MATERIALS AND METHODS
Virus Culture and Purification Tissue culture was maintained in accordance with established methods (Bird &
Forrester, 1981). Minimal essential media containing 5 °,% fetal calf serum (5%
DMEM) was used to grow Vero (CRL 1586) and Chinese hamster ovary (CHO) Kl (CCL 61 ) cells obtained from American - IU-Type Culture Collections. A 10 % suckling mouse brain (SMB) suspension of WEE
strain 71 V-1658 was kindly provided by Dr. Nick Karabatsos, Centers for Disease Control, Fort Collins, CO. WEE Fleming and California strains were purchased from ATCC (Mannanas, VA). WEE
B11 and CBA87 strains were kindly provided by Dr. (Jeorge Ludwig, United States Army Medical Research Institute of Infectious Disease (Frederick, MD). Seed stocks of WEE strains were made by inoculation of Vero cells with virus suspensions at a multiplicity of infection (MOI) of less than 0.1. For RNA isolation, virus stocks were prepared by infecting Vero cells at a MOI of 10. The virus was precipitated from cleared supernatant by the addition of polyethylene glycol MW 6000 to 7 %(w/v) and NaC',l to 2.3 %(w/v). It was subsequently purified on a 20-60 %(w/w) continuous sucrose gradient, followed by resuspension in PBS.
Nucleic Acid Preparation Viral RNA used in WEE strain 71 V-1658 library construction was prepared by the lysis of virus in 0.5 %(w/v) sodium dodecyl sulfate (SDS), and RNA extracted using the cesium chloride/guanidium isothiocyanate method previously described (Sambrook et al., 1989). RNA
was precipitated using sodium acetate; and ethanol, then stored at -70 °C. Prior to use, RNA was washed with 80 %(v/v) ethanol, dried and dissolved in nuclease-free water (Promega, Madison, WI). Integrity of the RNA was checked on formaldehyde agarose gels (Sambrook et al., 1989).
A cDNA library of WEE strain 71V-1658 was made by Invitrogen (San Diego, CA), by the ligation of cDNA into the BstXI site of prepared pcDNAII vector, and electroporation into electrocompetent DH1 F' Escheric~hia coli cells. Manipulation of RNA and DNA
followed established procedures (Sambrook et al., 1989; Ausubel et al., 1995). Rapid plasmid preparations were made using the Wizard' M plasmid purification kit (Promega, Madison, WI).
Large-scale plasmid preparations used the alkali lysi;s protocol as modified by Qiagen -Il-(Chatsworth, CA). For PCR, RT-PCIZ and DNA sequencing, oligonucleotide primer design was guided by information from WEE strain BFS1703 and other partially sequenced WEE strains (Hahn et al., 1988; Weaver et al., 1993), and from regions of sequence conservation (0u et al., 1982 &1983). Oligonucleotides were synthesized and gel purified either at the Regional DNA
Synthesis Laboratory (Calgary, Alberta), or on a Beckman Oligo 1000 DNA
synthesizer. A
catalog with the sequences of primers used is listed in Table 1.
Construction of pCXH 3 The Invitrogen WEE library was screened by dot. blot hybridization (Sambrook et al., 1989) with [3zP]-labeled, random primed RT-PCR fragments as probes (Amersham, Oakville, ON). A 3100 by insert, pcDW-12, vvas identified, and corresponded to the 3' end of the 26 S
RNA. The missing 5' end of the 26S region was generated by RT-PCR using the primers WEES'Sstl and WEEP3 (Table 1 ). The 1500 by SstIiNcoI restricted fragment was inserted into the plasmid, phT3T7BM+ (Boehringer Mannheim, Laval, PQ), to generate a XbaI
site on the 5' end. The 1500 by XbaIlNcoI fragment was excised, gel purified and inserted into the XbaI and NcoI restriction sites ofpcDW-12. 'The resulting clone, pcDWXH-7, encoded the complete 26S
region of WEE 71V-1658. The struictural gene insert from pcDWXH-7 was cloned into the mammalian expression vector, pCI (fromega, Madison, WI). The pcDWXH-7 plasmid was first linearized using.HindIII, followed by a Klenow fragment reaction to fill in the 5' overhang. The insert was then excised using XbaI, gel purified and ligated into the XbaIlSmaI digested pCI
vector. The isolated recombinant plasmid, pCXH-3, was characterized as having the correct insert by restriction mapping.
Construction of pVHX 6 The clone, pcDWXH-7, encoded the complete 26S region of WEE 71 V-1658 was digested with Sac I, and relegated in the reverse orientation. The isolate, pcDWHX-45, contained the complete 26S of WEE, with the reverse cloning sites (HindIII on the 5' end andXbaI on the 3' end). The WEE 26S gene segment was excised from pcDWHX-45, and cloned into the HincIIII
and XbaI sites of the mammalian ex~:~ression vector, pVAX (Invitrogen, La Jolla, CA). After transformation into E. coli DHlOa. (Life Sciences, Burlington, ON) and screening of inserts by restriction analysis, a resulting isolate, pVHX-6 was identified. SEQ ID NO: 3 shows the upstream portion of the pVHX-6 vector to where it becomes the XH-7 sequence.
The remaining nucleotide sequence of pVHX-6 from the point of divergence is identical to that of structural gene pcDWXH-7 of SEQ ID NO: ?.
Expression of the Structural Genes o f WEE
The pCXH-3 expression vector was transfected into Vero or CHO K1 cells using the cationic lipid, LipofectamineTM (Gibco/BRL, Burlington, ON). Briefly, Vero or CHO K1 cells were grown to 30-50 % confluency in Costar 6-well plates. The monolayers were transfected with pCXH-3 in accordance with the vmanufacturer's directions, for a period of 5 hrs, followed by a further 29 hr incubation after the addition of 5% DMEM. The monolayers were fixed in methanol:acetone (1:1 ) for 5 min and washed with PBS containing 0.1 %(v/v) Tween 20 and 3 BSA (PBS-TB). The cells were incubated 45 min at 37 °C'. with a 1/100 dilution (in PBS-TB) of concentrated cell supernatant from hybridoma cell lines expressing monoclonal antibodies to the WEE El (clone IlD2) or E2 (clone 3f3) proteins, followed by washing with PBS-TB.
Monolayers were incubated with a 114000 dilution of goat anti-mouse IgG/IgM (H
& L) horse radish peroxidase conjugate (Caltag, .So. San Francisco, C:A) for 45 min at 37 °C. After washing with PBS-T, 2 mL of TruBlue ~'~1 HRP substrate (Kirkegaard & Peny Laboratories, Gaitherburg, MD) was added, and plates were incubated a further 30 min at room temperature, followed by microscopic examination.
In a second method, one-step in vitro transcription and translation reactions using the TNT coupled system (Promega Corporation, Madison, WI) was used to express the gene S products from both pCXH-3 and pVHX-6, as both have an upstream T7 promoter which can be used for in vitro expression of inserts. The RNA was translated in the presence of [3sS]methionine to produce radiolabeled WEE proteins, which were further processed with canine pancreatic microsomal membranes. All connponents of the in vitro transcription and translation reactions were incubated together for 90 min at 30 °C.
Results were analyzed by SDS-PAGE or radioimmunoprecipitation.
Radioimmunoprecipitation The TNT reactions were dilutf;d to a volume of 500 ml with RIP buffer consisting of 0.1 S
M sodium chloride, 0.1 % SDS, 50 mlvl Tris-HC1 pH 7.4, and 1 % Triton X-100.
They were then preabsorbed by incubating with 75 J L of protein Ci-agarose (Gibco BRL) for 30 min at room temperature. The samples were centrifuged at 13,000 rpm for 1 min, and the supernatants were then immunoprecipitated with either ll 00 ~L of supernatants from anti-WEE
hybridoma cells or pg of purified anti-WEE antibodies. The reactions were incubated for 1.5 hr at room temperature, after which 75 pL of protein G-agarose was added. The reactions were incubated 20 for an additional 30 min at room temperature. Immunoprecipitated proteins were collected by centrifuging at 13,000 rpm for 1 min. The pellets were washed with S00 pL of RIP buffer and centrifuged at 13,000 rpm for 1 miry; 'this step was repeated three additional times. The pellets were resuspended in 2x Laemmli sample buffer (Bio-Rad Laboratories) containing fresh 2% b-mercaptoethanol and heated at 100 ''C" for 10 min. The samples were centrifuged at 13,000 rpm for 1 min, and the supernatants were collected. The immunoprecipitated [35S]labeled WEE
proteins were further analyzed by SDS-PAGE and autoradiography. Radiolabelled [~'~C]molecular weight markers from Amersham Pharrriacia Biotech were also run on the polyacrylamide gels.
DNA Sequencing Automated sequencing of the 26S region was performed using the ABI Prism Dye Terminator Cycle Sequencing or :Big-DyeTM Terminator Cycle Sequencing kits of plasmid templates according to the manufacturer's instructions (PE-Applied Biosystems, Foster City, CA). Sequencing reactions were purified on Centri-Sep ~ M columns (Princeton Separations, Adelphia, NJ), dried and analyzed on an ABI 373 or 310 automated sequencer.
For the nonstructural region, template cDN,As were generated in a single-step integrated RT-PCR
procedure using the TitanTM RT-PCR kit (Boehringer l~iannheim, Laval, PQ), following the manufacturer's suggested protocols. IRr-PCR products were purified using the QIAquick~~M PCR
Purification kit (Qiagen, Chatsworth, CA) and sequenced (50-100 ng DNA per reaction). The extreme 5' end of the genome was not sequenced in WEE 71 V-1658. However, a 5' RACE
reaction (Frohman et al., 1988) was used to obtain a cDNA fragment from the 5' terminus of WEE strain CBA87. Briefly, primer WEESS9 (GGTAGATTGATGTCGGTGCATGG) was used to prime reverse transcription of the :>' terminus of the viral RNA. After poly(A) tailing of the cDNA with terminal transferase, a plus sense primer (GTACTTGACTGACTGTTTTTTTTTTTTTTT) was used in conjunction with WEE559 for amplification of the 5' terminus.
Nucleotide Sequence Analysis and Assembly -IS-Sequence traces were edited manually and assembled using the Seqman component of the Lasergene DNA analysis software (DhtASTAR, Madison, WI). Codon preferences and patterns were assessed using the CodonUse and CodonFrequency programs, while the overall frequency of mononucleotide and dinucleotides was calculated using the Composition program (Wisconsin S Package, Version 9.0, Genetics Computer Group, Madison, WI). Quantitative assessments of sequence similarities (nucleotide and amino acid), were calculated by preliminary alignment using the Pileup program, followed by manual alignment adjustment, and analysis with the Distances program (GCG). Amino acid sequences aligned as described, were used as the basis for generating phylogenetic trees (GCG). The GeneQuest module of the Lasergene program (DNASTAR, Madison, WI) was used to predict and calculate RNA secondary structures at the ends of the genomic RNA using minimal energy calculations. Multiple sequence alignments were accomplished using the Clustal component of MegAlign (DNASTAR). The complete WEE
genomic nucleotide sequence has been submitted to GenI3ank (Accession Number AF 143811 ).
Administration of DNA or Inactivated Virus DNA solutions or an inactivated WEE virus vaccine in PBS, were administered to the mice by ballistic or intramuscular (IM) routes. For IM route of administration, a 27 g needle was used to deliver 50 ~g of DNA (pCx.H-3 or pCl - negative control) or 50 pL of inactivated WEE
vaccine (SALK WEE inactivated vaccine). 'The volume of inoculum used was 100 pL, diluted in PBS. Fifty pL was administered IM to each of the hind leg muscles of a mouse.
When boosters were given, they were administered 14-28 days apart. Fc~r ballistic administration, mice were shaved in the abdominal area with elc;ctric hair clippers. 'The mouse was subjected to ballistic delivery of DNA coated onto gold pat7:icles following the manufacturer's standard specifications.
The Helios Gene Gun (Biorad, Mississauga, ON) was used as directed, at a pressure setting of 400 psi. Mice were given 1.25 ~g DNA and 0.5 mg gold, 1 ~m diameter, per shot, and up to three shots for one dose time. Boostenrs were given 14-28 days apart. The mice were challenged 14-28 days after the final booster.
S Mouse Infectivity with WEE
Female BALB/c mice, 17~-2'_i g, were obtained from the mouse breeding colony at Defence Research Establishment Sul'field (DRES), with 'the original breeding pairs purchased from Charles River Canada (St. Constant, Quebec, Canada). The use of these animals was reviewed and approved by Animal C'.are Committee at DRES. Care and handling of the mice followed guidelines set out by the C.'anadian Council on Animal Care. Virus was administered to the mice by intranasal (IN) or intraperitoneal (IP) routes. The volumes of inoculum used were 50 pL for IN and 100 p.L for IP. For IN administration, mice were anaesthetized with sodium pentobarbital (50 mg/kg body weight, intraperitoneal). When the animals were unconscious, they were carefully supported by hands with their nose up, and the virus suspension in PBS was gently applied with a micropipette into the nostrils. The applied volume was naturally inhaled into the lungs. For IP infection, the mouse was manually restrained, and a 1 ml tuberculin syringe fitted with a 27 g needle was used to administer approximately 100 L~L of the virus suspension in PBS.
Infected animals were observed daily, for up to 14 days post infection.
RESULTS
Complete Nucleotide Sequence of WEE Genome and Deduced Amino Acids The nucleotide sequence of WEE strain 71 V-1658 (SEQ ID NO: 1) was determined via several distinct sequencing strategies, as summarized in Figure I . The 5' terminus of 25 nt was not determined for this strain. However, it was determined by sequencing a 5' RACE product -t~-from strain CBA87. Excluding the terminal 5' cap structure and the 3' poly(A) tail, the genomic sequence of WEE was found to be 11,508 bases long. The base composition was 28 % A, 25 C, 25 % G, and 22 % U. The dinucleotide usage of the WEE genome was compared with those values anticipated from the base composition. Several dinucleotides were found in lower S proportions than anticipated, notably UpA (81 %), CpG (8:3%) and CpC (85%) (data not shown).
Codons containing the CpG dinucleot ide were present at 82% of the anticipated value, including codons for serine (78%), proline (80~~°) and arginine (78°,%).
The WEE 71 V-1658 sequence was used to conduct a variety of phylogenetic analyses with previously determined alphavirus sequences. The alphaviruses used in the analyses included EEE strain North American variant (Genbank Acc. No. X67111), O'Nyong Nyong (ONN) strain Gulu (Genbank Acc. No. M33999), Ross River (RR) strain NB5092 (Genbank Acc. No. M20162), Semliki Forest (SFV) (Genbank Acc. No. J02361 ), SIN strain HR (Genbank Acc. No. J02363) and VEE ID (Genbank Acc. No. 1.,04653). The degree of conservation among the various sequences (nucleotide acid amino acid) through the stereotypical alphavirus genome is shown in Table 2. The carboxy-terminal domain ofd nsP3, which consistently fails to exhibit homology among sequenced alphaviruses, was excluded from this comparison as it has been adjusted for in previous analysis (Weaver et al., 1993). The deduced amino acid sequences for nsPl-4 of WEE 71 V-1658 demonstrated closest identity to the corresponding proteins from EEE
(Table 1 ), reflecting similar observations made for nsP2 and nsP3 of WEE 5614 and EEE
(Weaver et al., 1993).
Nontranslated Terminal Regions Alignment of the 5' terminal nucleotide sequences of WEE CBA87 and WEE 71 V-is shown in Figure 2a, along with a comparison of the 5' termini from EEE and VEE. 'the close _ ~g _ similarity between WEE and EEE, has been verified expE;rimentally, in that a EEE/Highlands J
degenerate primer, EHJS', was able to P(:R amplify the 5' end of the WEE
genome, while an analogous SIN primer could not (data not shown).
Potential stem loop structures were found in WEE 71 V-1658, including a stem loop at the extreme 5' terminus (2-30) and a pair of stem loops (13'7-189) (Figure 3a).
The homologous structures for EEE are also shown (Figure 3b) (0u et al., 1983). Minimal energy values calculated for the stem loops were similar between WEE and EEE. Further analysis ofthe region between the structures described above, indicated a large, highly base-paired stem loop structure (39-131 ), that had not been previously described, and was observed in SIN and EEE in a similar location (data not shown).
The sequence of WEE 71 V-1658 3' NTR, overall, shared little homology with any of the alphaviruses examined, but included the highly conserved 19 nt region at the 3' end ( 11490-11508), which was identical to that determined for WEE BFS 1703 by Hahn et al., 1988. Two copies of the characteristic 40 base Sindbis-like terniinal repeats as previously reported (0u et al., 1 S 1982) were found in WEE 71 V-1658 ( 11234-11273 and I 1292-11331 ).
However, the 3' NTR of WEE showed some surprising results that had not been previously described. The first 40 nt terminal repeat formed the backbone for the formation of a 57 nt double stem loop structure (11228-11284) (Figure 4b), consisting of an a and (3 loop. The second 40 nt repeat of WEE
formed a nearly identical 59 nt double stem loop structure ( 11285-11343), directly adjacent to the first structure. SIN with three 40 nt repeats, forms three double stem loops (Figure 4a) while EEE, which does not contain a SIN-like 40 nt repeat, contains the a and ~i loops (Figure 4c).
Nonstructural Region Comparisons within the nonstructural regions (4475 nt) of WEE strains 71 V-1658 and 5614 (Weaver et al., 1993), yielded 9~4 nt changes resulting in 26 amino acid substitutions (1.8%
difference) as summarized in Table 2. The most notable variation, a three-base deletion (4530) within the nsP3 gene of WEE ? 1 V-1 ti58 constitutes the only insertion/deletion observed within the polypeptide encoding regions. A short hypervariable region was observed (1421-1449), where 11 of 28 nt were different between the two WEE strains (Figure 2b). The presence of an opal termination codon and partial read-through site at the junction of nsP3 and nsP4 is consistent with WEE 5614. Extending previous phylogenetic analyses of WEE
(Weaver et al., 1993, 1997), phylogenetic trees depicting viral relatedness were constructed with the Distances program (GCG), for the unexamined genes (nsP 1, nsP4) and the entire nonstructural polypeptide encoding region (Figure 5). The data reveals the close relationship of WEE to EEE, relative to the other alphaviruses analyzed.
Structural Genes The largest WEE cDNA clone; isolated, pcDW-12, was 3100 by in size, but missing 5 nt and the poly(A) tract from the 3' end as determined by restriction mapping and DNS sequence analysis. The missing 5' 1500 by fragment was synthesized using PCR (primers WEES'Sstl and WEEP3) and subsequently cloned into pcDW-12 to yield a full-length clone of the structural genes (pcDWXH-7) (SEQ ID NO: 2;). Comparison of the: structural region of WEE

with WEE BFS 1703 (Hahn et al., I 988), indicated 53 nt changes, resulting in only 11 amino acid differences, of which two were nonconserved. One difference in residue was observed from the amino acid sequence of the N-terminus of the E2 protein of the WEE MacMillan strain (Bell et al., 1983), when this was compared to the deduced protein sequence of 71 V-1658. A short fragment (802 nucleotides) of the WEE 71 V-1658 E 1 protein gene, and the 3' NTR had been _?p_ published previously (Weaver et al., 1997); comparison with the sequence reported herein indicated no differences.
Expression of Structural Gene Expression of the insert from the cytomegalovirus (CMV) promoter was accomplished by transfection of the pCXH-3 plasmid into either Vero or CHO K1 cells. Cells expressing the E1 or E2 proteins were detected through the use of specific E1 or E2 monoclonal antibodies to WEE, followed by histochemical staining with the HRP substrate, Tru-Blue as demonstrated in Figure 6a. The control cells transfected with pCI alone showed no staining (Figure 6b), thus, demonstrating the fidelity of the proteins translated from the cloned 26S
region. In vitro translation of the insert using TNT T7 rabbit reticulysate and canine microsome system demonstrated synthesis of 'SS-metluonine-labelled proteins of the correct size as indicated by immunoprecipitation with monoclonal antibodies to the NC, E1 and E2 proteins (data not shown). Similarly, the construct pVHX-6 was along demonstrated to produce the correct MW
proteins as determined by in vitro transcription/translation. The level of expression for pVHX-6 was significantly higher then for pC:XH-3 (Figure 7).
Protection Against WEE Infection using DNA Immunization Different strains of WEE were shown vary in their virulence in BALB/c mice.
When similar amounts of WEE were given intranasally to BALB!c mice, time to death varied from 4 to 8 days. The California and Fleming strains were the most virulent (:Figure 8), and the Fleming strain was chosen as the challenge strain in protection studies. IP
administration of the virus did not kill adult mice (data not shown). Intramuscular administration of pCXH-3 did not show any protection, using one or two doses of 50 pg, followed by challenge 30 to 90 days after the final dose (data not shown). Intramuscular administration did result in an increase in antibody titre to WEE as determined by ELISA using a monoclonal antibody to the E 1 protein of WEE (data not shown). Expression and protection o f pCXH-3 DNA when delivered ballistically.
pCI was used as a control DNA. When two doses of pCXH-3 was given, protection of 50% was demonstrated as compared to no protection for pC;I (Figure 9) or PBS controls (data not shown). IM injection showed marginal protection (one group 25% survival - data not shown). The dose of WEE
Fleming strain (challenge strain) was 1.25 x 10'~ PFU for 100% killing via an intranasal route of infection. Preliminary studies examining protection using the pVHX-6 vector, indicated promise with this construct using the Gene Ciun, and ballistic delivery. With the pVHX-6 vector, one mouse succumbed immediately to tl~e effects of the sodium pentabarbital (anaesthetic). The remaining three mice showed no signs of coming down with a WEE infection, and remained completely heathy (Figure 10). Of the four pVAX control mice, all showed signs on WEE
infection, and two of the four mice died, while two did recover. A repeat of this experiment using 3 or 4 doses of pVHX-6, given 2 weeks apart, showed complete protection of the mice, similar to 3 doses of WEE inactivated vaccine (Figure 11 ). Three or 4 doses of pVAX showed results similar to the saline control, with only about 60% of the mice surviving Figure 11.
DISCUSSION
The WEE 71V-1658 genonnic: sequence of 11,508 bases was determined directly from cDNA clones of WEE or via sequencing RT-PCR products. fhe first 25 bases of the WEE
genome was determined indirectly, tlorough the use of a 5' RACE reaction in WEE CBA87.
Noting the relatively high conservation in the WEE sequences overall (1.7%
divergence) and in the overlap region between the two W EE sequences (see Figure 2a), it appears that the 5' ends of 71 V-1658 and CBA87 are of similar size and sequence.
_~»_ Comparison of WEE 71 V- l 658 to other partial sequences of WEE (Hahn et al., 1988;
Weaver et al., 1993) suggests little variation at the nucleotide level among these viruses (Table 2), showing an overall nt sequence difference of 1.7 '% over 8624 nt. Given a calculated rate of divergence of 0.028 % per year for the WEE E1 protein (Weaver et al., 1997), the expected nt S divergence for a difference in isolation of 18 years between the strains, should be O.S % (71 V-1658 isolated in 1971 and BFS 170~i in 195 3). The E 1 protein itself showed a rate of divergence of 1.S% in nt sequence between 71 V-1658 and BFS 1703. The lower rate observed by Weaver et al., (1997) could be due to greater conservation of structure at the C
terminus of E1, from where the rates of divergence were calculated. Areas with high rates of divergence were observed between WEE strains 71 V-16S 8 and :1614 at the 3' end of nsP 1 and the Send of nsP4 (Table 2).
The relatively high interstrain value for nsPl (4.S% difference) may be due to the presence of a small hypervariable region, with 11 of 28 nt changed in strain 5614 (Figure 2b). Variation in nsP4 occurred in a stretch of 21 nt at the 3' end of the Sfil4 sequence, and were left out of subsequent homology comparisons (similarity with the EEE sequence was maintained in this region). Discounting the carboxy-terminal region of nsP3 also gives a more accurate picture of the homology of the nsPl-4 nonstructural region (Weaver et al., 1993). The results for comparison of nt and protein sequences of WEE to other alphaviruses is shown in Table 2, and are similar to those obtained with nsP2 and nsP3 of 5614, when compared to other alphavirus sequences. Phylogenetic analysis (>f the WEE 71V-1658 deduced protein sequences of nsPl, nsP4 and the nsPl-4 region, as related to other alphaviruses (Figure S), illustrates the close relationship to EEE (HJ sequences were very limited for comparative purposes and were not included).
Assessments of codon usage frequencies and the frequency at which certain dinucleotides are found throughout the genome identified a number of statistical anomalies.
The slight CpG

dinucleotide deficiency previously described within other alphaviruses, and WEE itself, was confirmed in this study, at levels comparable to those reported (Weaver et al., 1993). The CpG
under representation is a typical feature of vertebrate genomes, and is not seen in invertebrates.
Viruses which infect dual hosts, such as the arboviruses, might be expected to utilize an intermediate nucleotide bias, as indicated by the slight CpG under-utilization observed in alphaviruses (Weaver et al., 1993). A pronounced under-representation of two other dinucleotides was also observed within the WEE genome, UpA, and CpC, a phenomenon noted throughout the genome, though thf; role of these codon preferences is unclear.
The 5' NTR sequence of WE:I? shows a close phylogenetic affiliation to EEE, and to HJ, although the HJ sequence information is more limited. 0u et al., {1983) had previouslypredicted (based on minimal free energy calculations) two hairpin structures at the 5' NTR of several alphaviruses including SIN and EEE. Both structures are present in WEE, the first ofwhich is a 5' terminal hairpin structure (2-30), similar to that calculated for EEE
(Figures 3a and b). The second is a dual hairpin structure (13 i-162, 16S-189) which is almost identical to that identified 1 S for EEE. The region between the tf;rminal and dual hairpins can itself form a long hairpin structure, and includes highly conserved stretches of 92 nt (data not shown).
The significance of these structures is currently unknown.
Previous reports (Hahn et al., 1988; Pfeffer et al., 1998) suggested WEE virus arose as a result of two recombination events between alphavirus-like ancestral viruses.
The first recombination occurred near the junction of the E3 and capsid genes. The second recombination occurred 80 nucleotides from the a' end of the genome. Evidence for the occurrence of the second recombination event is infewed from sequence similarities of the 3' NTR
between WEE, EEE and SIN, in which WEE shows greater similarity to F:EE (65 %) than to SIN
(50 ~%) in the last 100 nt of the 3' end. However, the apparent plasticity of the 3' NTR may only be reflecting _p4_ the selective pressures under which the nascent WEE virus evolved, resulting in rapid selection of 3' sequences which are more similar to EEE, and may not represent an actual recombination event as previously postulated.
The 3' NTRs of alphaviruses are characterized by widespread sequence divergence and yet contain small, strongly conserved motifs (reviewed in Strauss & Strauss, 1994; Pfeffer et al., 1998). Analysis of the 3' NTR indicated the presence of double stem loop structures among SIN
and WEE (Figures 4a and b). Interestingly, the 40 by repeat found in SIN and WEE is contained within the double stem loop structure,. SIN was found to contain 3 double stem loop structures and WEE was found to contain two. In SIN, the spacing between the three double stem loop structures was around 30 nucleotides, while in WE.E the distance was zero nt separating the structures. Additional alphaviruses were assessed and it is interesting to note that double stem loop structures were found in many of the WEE- and SIN-related viruses (SIN, Aura, Babanki, Ockelbo, Kyzylagach, Whataroa, WEE and HJ). The double stem loop structures found in SIN
and WEE viruses consisted of the a loop (ALJGUA[U/C]UU ) and the [3 loop (GCAUAAU) (Figure 4b). Surprisingly, while EEE does not have the 40 by repeat element found in SIN and WEE, it contains the a and [3 loop structures (Figure 4c). The significance of these conserved loop structures between SIN, WEE and EEI? viruses has yet to be elucidated, although previous studies suggest a role in viral replication and/or host specificity (Kuhn et al., 1990; Kuhn et al., 1991 ). For example a deletion of' 2.6-318 nt from 3' end of SIN, resulted in reduced viral replication in mosquito cells but not in chicken cells (Kuhn et al., 1990). In contrast, substitution of the SIN 3' NTR with the substantially different RR 3' NTR (which lacks the 40 by repeat and double stem loop structures), had nc:~ effect on the growth of the chimeric virus in mosquito cells, suggesting that host proteins interact with the 3' NTRs to cause differential host effects (Kuhn et al., I 991 ).
-ps-The 26S region of 71 V-1658 was placed under the control of the CMV promoter of pCI.
To test for functional expression of the pCXH-3 vector and for a functional product in cell culture, the pCXH-3 vector was transiently transfected into Vero cells. WEE
proteins were detected on the cell using specific monoclonal antibodies to both the E 1 (Figure 6a) and E2 proteins (data not shown). The binding specificity of these monoclonals has been previously determined by western blot analysis and immunoprecipitation analysis (data not shown). The use of pCXH-3 in DNA immunization experiments indicated that the construct could partially protect against WEE intranasal challenge using ballistic delivery. Preliminary results do indicate that WEE reactive antibodies can be detected by ELISA when the pCXH-3 plasmid is given intramuscularly (unpublished results). However, this afforded no protection to the mice, as there were no survivors. Intranasal (data not shown) delivery of the pCXH-3, with and without liposome encapsulation did not demonstrate any protection under the conditions used. Mice immunized with the pCI control plasmid did not show any signs of protection in these studies.
Expression of the WEE structural proteins in the pCI-based vector (pCHX-3) gave moderate to poor levels of expression in vitro, using the TNT' expression kit.
A new vector, pVAX (Invitrogen) was designed for L~NA immunization and was basically the same as pCI, but lacked the intron found in the pCI ve~~tor. Initial restriction mapping of pCXH-3 indicated the plasmid was the expected size, but later analysis indicated a extra 4 kb fragment was present (data not shown). The WEE structural proteins were cloned and expressed in pVHX-6, indicating the correct sized proteins by SDS-PAGE, and producing higher levels of WEE product in vitro (Figure 7). Preliminary results with pVHX-6 indicated it could completely protect mice against an intranasal challenge of WEE. While 50% of the pVAX mice did survive, they all demonstrated at least moderate to se;vf;re infection with WEE. It is possible that pVAX contains CpG motifs that show some protective; effect, through a nonspecific adjuvant like effect (Kreig et _-y_ al, 1998). However, there was a dramatic difference between the pVAX and the pVHX-6 group, in the protection afforded the two groups of mice.
The plasmids, pCXH-3 and pVHX-6 show promise as vaccine candidates for WEE.
This is especially important for protectic:>n against an aerosol challenge of WEE, and event that would be envisioned in a potential biological warfare attack using WEE as a biological warfare agent.
This agent is difficult to protect against if delivered aerosolly, as the agent is purported to travel up the nerves directly into the brain. The research is applicable to VEE and EEE, as these viruses can also cause encephalitis following a similar route of infection (equines and potentially human).
It is to be understood that the embodiments and variations shown and described herein are merely illustrative of the principles of this invention and that various modifications may be implemented by those skilled in the art without departing from the scope and spirit of the W vention.

Table 1. WEE 26S Region Primers Name Length Sequence WEE3' 30 GTAGTGTATATTAGAGACCCATAGTGAGTC

WEES'SST 20 'rCCAGATACGAGCTC.'ATACT

WEEN 1 20 GG'I'GC',CG(~T'GG,AGGCCGTTT

WEEN1A 20 GA'I'CTTAGGAGGTC'GATAGC

WEEN2 20 GGC',TGATC~AAACCACTCCAC

WEEN3 20 (. CACC'C GTGTGC'I'ATTCACT

WEEN3A 20 CGC".CG'CGTTTC.~rGCCCAATA

WEENS 20 GGC~:ATCAC:CCTCCACCTGAC

WEEN7 20 CTA'CTGATCATGCAG TCGCA

WEEN8 20 a~GTGGAG(:C.'TC'CGC'C_~AGCGT

WEEN9 20 GAGGAGTGCiGCGGGAAAGGC

WEEN12 20 ACTGTCATTC1T(~C'.TGTGTGG

WEEN14 20 CG'hCATCAGAAAGGGGCTTG

WEEN I6 20 GGAAAGCTC~GT'AAACiTGCCA

WEENO 20 GGAGAAC(~ACATAAAGTCGA

WNSP 1 25 (iGC."rAACGTGGACAGGGACGTGATG

WEEPO 20 GGC','rA'rCGACC'l'CCTAACrAT

WEEPOA 20 CTGTCGGTTC.'CCTGGTTTAG

WEEP2 20 CGTTCTCCAC iCACiCGTGTCG

WEEP2A 20 1'ATTGGGCTGAAACACGGCG

WEEP3 20 C'TTCAAGT G AT( "C~TAAACGT

WEEP4 20 rICTCCAGCCCT'~'C'.TCGCCCC

WEEPS 20 C~TTC".GACCAACC1CCTTATAC

WEEP7 20 C~G'rGA~I'T(~TGA'I~'CrA'1'CTCAC

WEEP10 20 (.'CT'I'GAT(:~T(~'ATGG'TCGTGG

WEEPI 1 20 TG<:'ACTGAGTGCrTCTCiTGTG

WEEP12 20 ATCiTTrC'CAGC'G7'TGGTTGGC

WEEPl 3 20 C;TGTTCTCAC'. TCTTCACAGAA

WEEP14 20 ATG'T'G'I'GC~TCGC'TTCCTTCA

_pg_ Table 2. Percentage Variation in Nucleotide and Encoded Amino Acid Sequences Between WEE 71V-1658 and Other Alphaviruses WEE WEE EEE VEE SIN RR ONN SF
(BFS1703) (5614) 5' NTR - -nsPl (nt) - (4.5) 25.1 34.8 40.9 37.8 39.7 39.1 nsPl (aa) - (6.3) 15.1 32.1 40.3 35.5 37.2 33.3 nsP2 (nt) - 1.8 28.2 34.6 43.9 42.1 42.9 42.8 nsP2 (aa) - 0.(i 16.2 2(i.5 44.9 43.2 44.9 44.4 nsP3 (nt)* - 1.8 30.2 36.7 45.8 39.3 42.6 42.2 nsP3 (aa)* - 2.1 18.8 32.4 46.3 38.7 40.9 43.5 nsP4 (nt) (1.8) (2.4) 25.6 31.4 34.7 35.3 36.0 37.0 nsP4 (aa) (2.6) (4.3) 11.7 21.4 26.8 27.3 25.8 27.4 Intervening 4.3 - 56.6 ~ 1.5 47.6 44.7 60.0 47.7 (nt) Capsid (nt) 2.1 - 26.3 40.8 47.7 46.3 47.5 48.2 Capsid (aa) 1.5 - 16.8 43.5 52.8 53.3 54.6 54.3 E3 (nt) 1.1 - 45.6 40.7 38.3 51.7 47.5 46.7 E3 (aa) 1.7 - 38.0 39.6 39.4 46.0 45.8 43.9 E2 (nt) 1.2 ~- 51.2 52.3 36.2 51.7 55.3 52.8 E2 (aa) 1.0 -- 59.0 60.0 31.7 63.5 65.7 64.7 6K (nt) 0.6 ~- 53.3 46.3 26.1 51.9 50.3 54.3 6K (aa) 1.8 -- 65.6 59.3 32.7 72.2 69.1 75.9 E1 (nt) 1.5 ~- 43.8 45.8 29.6 47.2 48.5 44.4 E1 (aa) O.S -- 49.0 51.0 23.4 51.5 54.8 50.3 3' NTR (nt) 0.7 -- 57.8 55.0 53.2 69.1 65.8 60.3 * based on rminal N te domain, C terrrunal domain dis<;arded due to lack of homology between alphaviruses ( ) based mplete (289 207 BFS1703, on inco sequence nt) nt 113 data: and for nt nsPl nsP4 for ( 5614) - no data _09_ Canadian Seq-1416-l5ver2 rev apr 15 2002 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: The Minister of National Defence, Government of Canada (ii) TITLE OF INVENTION: Novel DNA-Based Vaccine Against the Encephalitis Alphaviruses (iii) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS:
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(A) APPLICATION NUMBER: CA 2,327,189 (B) FILING DATE: 2000/12/Z1 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Nelligan 0'Brien Payne LLP
(C) REFERENCE/DOCKET NUMBER: 14792-4 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11484 (B) TYPE: nucleic acid (C) STRANDEDNE55: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: western equine encephalomyelitis virus -strain 71v-1658 (vii) IMMEDIATE SOURCE: Western equine encephalomyelitis virus Viruses;
ssRNA positive-strand viruses, no DNA stage;
Togaviridae; Alphavirus (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 25 .. 7428 (D) OTHER INFORMATION: 5' UTR <1 .. 24 (x) PUBLICATION INFORMATION:
(A) AUTHORS: Netolitzky,D.J., Schmaltz,F.L., Parker,M.D., Rayner,G.A., Fisher,G.R., Trent,D.W., Bader,D.E. and Na~ata,L.P.
(B) TITLE: Complete genomic RNA sequence of western equine encephalitis virus and expression of the structural genes (C) JOURNAL: J. Gen. virol.
(D) VOLUME: 81 (E) ISSUE: Pt 1 (F) PAGES: 151 - 159 (G) DATE: 2000 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
accctacaaa ctaatcgatc caatatggaa agaattcacg ttgacttaga tgctgacagc 60 ccgtatgtca agtcgttaca gcggacgttt ccacaatttg agatcgaagc aaggcaggtc 120 Canadian -1416-l5ver2 5eq rev apr actgacaatgaccatgccaatgccagagcgttttcgcatgtggcaacaaagctcattgag 180 agcgaagtcgaccgggaccaagttatcttggacattggaagtgcgcccgtcagacatgca 240 cattccaatcaccgctatcattgtatctgccctatgataagcgctgaagacccggacaga 300 ctacaacggtatgcagaaagacttaagaaaagtgacattaccgacaagaacatagcctct 360 aaggcggcagacctgctggaagtcatgtcaacaccagacgcagagactccatctctgtgt 420 atgcacacagacgccacgtgtaggtactttggaagtgtagcagtataccaagatgtgtac 480 gcagtccatgcaccgacatcaatctaccaccaggcgcttaaaggagttaggacaatttac 540 tggataggctttgacacgaccccttttatgtacaaaaacatggcaggttcctaccctact 600 tacaacacgaactgggctgacgagagagtattggaagcacgtaacattggcctcggtaac 660 tcagatcttcaggagagcaggcttggaaaactctcaatccttaggaagaagaggctccaa 720 cctactaataagatcatattctcggttggttcaacaatctacacagaagatagatcactg 780 ttacgtagctggcatcttccaaacgtgttccacttgaaaggaaagtctaacttcacaggt 840 agatgtgggaccattgtcagctgtgaagggtacgtcatcaaaaagataacgatcagccca 900 ggactatacggtaaagttgagaacttggcgtccacaatgcatcgcgagggtttcttgagt 960 tgcaaagtcacagatacgctgcgcggcgagagggtttcttttgctgtgtgtacgtatgta 1020 ccagccacactttgcgatcagatgacagggattctggcaactgacgttagtgtggatgac 1080 gcacaaaaactattggttgggctcaaccaaaggattgtcgtcaatggtaggacgcaaaga 1140 aatactaacacaatgcagaactatctattaccagtggtcgcccaggcgttttccaggtgg 1200 gcgcgtgaacatcgtgccgacttggacgacgagaaagaactaggggtgcgggagcgcact 1260 cttactatgggctgctgctgggctttcaagacccagaaaatcacatccatctacaagaag 1320 cctggtacgcaaacaattaagaaagtacctgccgtctttgactcatttgtgattccacgc 1380 cttaccagccacgggctcgatatgggcttccgccgtaggctcaagctgctgcttgaacca 1440 actgtcaaacccgcaccggctattacaatggccgatgtggagcatctgcgtggcttacag 1500 caagaagctgaagaagtggctgcagcggaagagatcagagaagccctgccacccttgctc 1560 cctgaaatagaaaaagagaccgtagaggcagaagtagacctcattatgcaagaggcagga 1620 gcaggtagcgtggagacaccacgaggacacatcagggtgacaagttacccaggcgaagag 1680 aagattgggtcttacgctatactttcaccccaggcggtattgaatagtgaaaaactggcg 1740 tgtatccacccattggcggaacaagtactggtaatgactcacaaaggtagggcagggaga 1800 tacaaagtcgagccataccacggtaaggtcattgtaccagaagggacggcggtccctgtt 1860 caagacttccaggcattgagtgagagcgctacgatcgttttcaacgagagggagttcgta 1920 aacagatacctgcaccacatcgcaatcaacggaggagcgctaaacactgacgaagagtac 1980 tataagactgtaaagactcaggacacagactcagaatacgtcttcgatattgacgcacga 2040 aagtgtgttaagcgagaagacgcaggtcccttgtgcctaaccggtgatctggtagatcca 2100 ccatttcacgagtttgcgtacgagagtctcaagacacgaccagcagcacctcacaaagtc 2160 ccaaccatcggagtctatggagtgccaggttcaggtaaatctggaatcatcaaaagcgct 2220 gtgactaagaaagatctggttgtgagtgcgaagaaggaaaactgcgcagaaatcatcagg 2280 gatgtaaggaggatgagacgtatggatgttgctgctaggactgtcgattcagtgcttcta 2340 aatggggttaagcaccccgttaacactctgtacattgatgaggcatttgcctgccatgca 2400 gggacgctgctggcactgattgccatcgtcaaacctaagaaagtggtattgtgcggggac 2460 ccaaaacaatgcggcttctttaacatgatgtgcctgaaagtacattttaaccatgacata 2520 tgcactgaagtgtaccataaaagcatctctaggaggtgcacacagactgtaaccgccatc 2580 gtctccacgctcttctacgacaagcgaatgaagacggttaacccatgtgctgataaaatc 2640 atcatagataccacagggaccacaaagccgcacaaagatgatctgattctaacctgtttc 2700 agaggatgggtgaaacagctacagattgactacaaaaatcacgaaatcatgactgcggct 2760 gcatcgcaaggacttacgcggaaaggcgtttatgctgtcaggtacaaagtcaacgagaat 2820 ccactctactcgcagacttctgagcacgtgaacgtgttacttacacgcacagaaaaacgc 2880 attgtctggaagacgctagctggtgatccctggataaagacacttacagctaaatatccc 2940 ggggatttcacggcttcattggacgactggcagcgcgaacacgacgccattatggcacgc 3000 gttcttgataagccgcagacagctgatgtgttccagaataaggtgaacgtctgctgggcg 3060 aaggctttagagccagtcttggccacggccaacattgtgctgacgagacagcagtgggag 3120 acgttgcacccattcaagcatgacagagcgtactcacctgaaatggcactgaacttcttt 3180 tgcaccaggttctttggagtagacctggacagtgggttattttccgctcctaccgtcgca 3240 cttacttacagggatcagcactgggataactcgccagggaagaacatgtatgggcttaat 3300 agagaggtagcaaaggagttgtcacggcgatatccgtgcatcacaaaagcggttgacaca 3360 ggcagggtagctgatataaggaataataccatcaaggactactctccaacaattaatgtg 3420 gttccattaaatcgccggttgccccactcgttgatcgttgaccacaaaggacagggtaca 3480 actgatcacagcggattcctatctaagatgaagggcaaatctgtgttggtgatcggcgat 3540 cctatcagcattccagggaagaaagtagagtccatgggtccattgcccactaataccatc 3600 aggtgtgatctcgatttgggaatacctagccatgtcggtaaatatgacattatctttgtc 3660 aatgttaggaccccgtacaggaaccatcactaccaacagtgcgaggatcacgctatccac 3720 cacagcatgctaacgtgtaaggctgtccaccacctgaacactggcggaacatgtgtggct 3780 atagggtatgggcttgctgatcgcgcaaccgagaatatcatcactgcggtggcacgctca 3840 tttaggtttacccgtgtctgtcagcctaagaacactgccgaaaatactgaggttctcttc 3900 gtgttcttcggcaaggacaacggcaaccacacacatgaccaggacagactcggtgtagtg 3960 cttgacaacatctatcaagggtcaaccaggtacgaggcagggagagctccagcgtacaga 4020 gtgatcagaggtgacattagcaagagcgctgaccaagctatcgttaatgctgctaatagc 4080 aaaggtcaaccaggttccggagtgtgcggtgcactgtaccgaaaatggccggctgctttt 4140 gatagacagccaatagctgtcgggacggctagacttgtgaagcacgaaccgctcatcata 4200 Canadian -1416-l5ver2 5eq rev apr catgctgtaggacccaatttttctaagatgccggaaccggagggcgaccttaagctcgca 4260 gctgcctacatgagcatagcgtccatcgtcaacgctgagcggattacaaaaatatcagta 4320 ccgctactgtcaaccggcatctattctggtggcaaagatcgagtgatgcaatcattgcat 4380 cacctgttcactgctttcgacactacggatgccgatgtcaccatatattgcttggataaa 4440 caatgggagaccaggataatcgaggccattcaccgcaaagaaagcgtcgaaattctggat 4500 gatgacaagccagtagacattgacttggtcagggtccacccaaacagctctttggcaggc 4560 agaccaggttactccgtcaatgagggcaagttgtattcatacctggaaggtacacgattc 4620 catcagaccgccaaggacattgccgaaatccatgcaatgtggcccaacaaatctgaggct 4680 aatgagcagatttgcttgtacatcctgggggagagtatgtccagcatccgctccaaatgc 4740 ccagtagaggagtcagaggcgtctgctccacctcacacacttccatgcctgtgtaattac 4800 gctatgacggctgagcgcgtatacaggttgcgctctgcgaagaaagaacagttcgccgta 4860 tgctcatcattcctgttgccgaagtacaggatcacaggcgtgcagaagctacagtgcagc 4920 aaaccagtcctgttttcaggcgtcgtaccaccggctgtacaccccaggaagtacgcggaa 4980 ataattctagaaacgccaccaccgccagcaacgacaaccgtaatatgtgaaccaactgtg 5040 ccagaacgtatacccagtccggtgatttctagagcaccaagtgcggaatcactgctatcg 5100 cttggcggcgtctcgttctctagctctgccacacgctcgtcaaccgcctggagcgactat 5160 gacaggcggtttgtggttacagctgatgtgcatcaagcgaacacgtctacgtggagcatc 5220 cctagtgctcctggcttggacgtccagctgccttctgacgtcactgattcccactggagt 5280 attccaagtgcatcaggctttgaagtgagaacaccatctgtacaggacctaactgcggag 5340 tgtgcgaagcctcgtggactggccgaaataatgcaagacttcaatactgctcctttccag 5400 tttctttcggactacagaccagtaccggcaccacggagacgccccatcccatcacctaga 5460 tcgacggcttccgcacctccagttccaaagccacgcaggactaagtaccaacaaccacca 5520 ggagtcgctagagcgatctcagaagcggagttggacgagtacatccgtcaacactccaac 5580 tgacggtatgaagcgggagcgtatattttctcatcggaaacaggccaaggtcaccttcaa 5640 cagaaatcagtacgtcaatgtaaactacaagaacctatattggatcgggccgtccatgag 5700 aagtattacgccccgcgcctcgatctcgaaagagagaaaatgttacagaagaaactgcaa 5760 ttatgcgcctctgaaggaaatagaagcaggtatcaatcacgaaaagtagaaaatatgaaa 5820 gcaattacagcggagcgactcatttctggattgggcacatatctatcatcagaagtgaat 5880 cctgtcgagtgttacagagtcaattatcctgtaccaatctactcgtcaacggtaattaac 5940 aggtttacatctgcagaggtcgcggttaaaacgtgcaacttagttatccaagagaattac 6000 cctacagtagccagttattgtataacagatgaatacgatgcgtatcttgacatggtggac 6060 ggcgcatcgtgctgtctagatacagccactttttgtccggctaaactgagaagctaccca 6120 aagaagcatagctatttgcagccagagataagatcagccgtcccatcgcctatacagaat 6180 acattacaaaatgtattggctgcagctactaaaaggaattgcaacgttacccaaatgcga 6240 gaattacctgtcttagattcggcggcatttaatgttgattgtttcaagaaatacgcatgc 6300 aatgatgagtactgggatacctttcgcgataaccctattcggctaactacagagaacgtt 6360 acgcaatatgtgacaaagctgaaagggccgaaagcagcagcattgtttgcgaatactcat 6420 aatctaaaaccgttgcaggagataccaatggatcaattcgtcatggatctaaagagagat 6480 gtcaaagttactcccggcacgaaacatacagaggagcggcctaaggtgcaggttattcag 6540 gctgcagatccccttgctaccgcttacctttgcgggatccatcgggaattagtccgtaga 6600 ctgaatgcggtgcttctgccaaatatccatactctcttcgacatgtcagcggaagatttt 6660 gatgcgattattgctgaacatttccaccacggcgacccagtattggaaacggacatcgcg 6720 tcgtttgataaaagcgaagacgacgctatcgccatttcggcgttgatgatccttgaggac 6780 ttaggtgtcgaccaaccgctcttagatttgatagaggcggcgttcggcaatatcacatct 6840 gtgcacctacctacaggaacgaggtttaaatttggtgccatgatgaaatccggtatgttc 6900 ttaacgctgtttgtcaacacactagtcaatatcatgattgctagcagagtactacgtgaa 6960 cggttaaccacgtcagcgtgcgcggcctctatcggcgacgataacatagtgcatggtgtc 7020 gtctccgacaccttgatggcggagagatgcgccacttggctgaacatggaagtaaaaatt 7080 attgatgcagttattggtatcaaagcaccctacttctgtgggggatttatcctggtggac 7140 cagataacaggcacagcctgcagagtcgcagaccctctaaaaaggctttttaagcttgga 7200 aaaccattgccagtcgatgatacccaagactgcgaccgccgccgggcactgcatgatgaa 7260 gcaatgcgatggaacagaattggaattacggacgagttagtgaaggccgtagaatccaga 7320 tacgagatcatactggcaggcctgatcatcacgtctctgtccacgttagccgaaagcgtt 7380 aagaacttcaagagcataagagggagcccaatcaccctctacggctgacctaaataggtg 7440 acgtagtagacacgcacctacccaccggcagaatgtttccataccctcagctgaactttc 7500 caccagtttaccctacaaatccgatggcttaccgagatccaaaccctcctaggcgccgct 7560 ggaggccgtttcggcccccgctggctgctcaaatcgaagatcttaggaggtcgatagtca 7620 acttgactttcaaacaacgatcacctaatccgccgccaggtccaccgccaaagaagaaga 7680 agagtgctcctaagccaaaacctactcagcctaaaaagaagaagcagcaagccaagagga 7740 cgaaacgcaagcctaaaccagggaaacgacaacgtatgtgtatgaagttggagtcggaca 7800 agacatttccgatcatgctgaacggccaagtgaatggatatgcctgcgttgtcggaggaa 7860 ggctgatgaaaccactccacgttgaaggaaaaattgataatgagcaattagcggccgtga 7920 aattgaagaaggctagcatgtacgacttggagtacggcgacgttccccagaacatgaaat 7980 cagacacgctgcagtacaccagcgacaaaccaccgggcttctacaactggcaccacggcg 8040 cagtccagtatgagaatgggagatttaccgtaccgagaggagtgggcgggaaaggcgaca 8100 gcggaagaccgatcctggacaacagaggcagagttgtggctattgttctaggaggtgcaa 8160 atgagggcacgcgtacggcgctttcagtggtcacttggaaccagaaaggggtgaccatta 8220 gggatacccccgaaggttctgaaccgtggtcactagttacagcgctatgcgtgctttcga 8280 Canadian -1416-l5ver2 seq rev apr atgtcacgttcccatgcgacaaaccacccgtgtgctattcactgacgccagaacgaacac 8340 tcgacgtgctcgaagagaacgtcgacaatccaaattacgacacgctgctggagaacgtct 8400 tgaaatgtccatcacgccggcccaaacgaagcattaccgatgacttcacactgaccagtc 8460 cctacctggggttctgcccgtattgcagacactcaacgccgtgtttcagcccaataaaaa 8520 ttgagaacgtgtgggacgaatctgatgatggatcgattagaatccaggtctcggcacaat 8580 tcggctacaatcaggcaggcactgcggatgtcaccaaattccgttacatgtctttcgacc 8640 acgaccatgacatcaaggaagacagtatggagaaaatagctatcagcacatctggaccct 8700 gccgtcgtcttggccacaaagggtacttcctgttagctcaatgtcctccaggtgacagtg 8760 taaccgtcagtatcacgagcggagcatctgagaattcatgcaccgtggagaaaaagatca 8820 ggaggaagtttgtcggtagagaggagtacttgttcccacccgtccatggaaagctggtaa 8880 agtgccacgtttacgatcacttgaaggagacgtctgccgggtacataaccatgcacaggc 8940 caggcccacacgcgtataagtcctatctggaggaagcgtcaggcgaagtgtacattaaac 9000 caccttctggcaagaacgtcacctacgaatgtaagtgtggcgactacagcacaggtatcg 9060 tgagcacgcgaacgaagatgaacggctgcactaaagcaaaacagtgcattgcctacaaga 9120 gcgaccaaacgaaatgggtcttcaactcgccggatcttattaggcacacagaccactcag 9180 tgcaaggtaaattgcacattccattccgcttgacaccgacagtctgcccggttccgttag 9240 ctcacacgcctacagtcacgaagtggttcaaaggcatcaccctccacctgactgcaatgc 9300 gaccaacattgctgacaacgagaaaattggggctgcgagcagacgcaacagcagaatgga 9360 ttacagggtctacatccaggaatttttctgtggggcgagaagggctggagtacgtatggg 9420 gtaaccatgaaccagtcagagtctgggcccaggagtcggcaccaggcgacccacatggat 9480 ggccgcatgagatcatcatccactattatcatcggcatccagtctacactgtcattgtgc 9540 tgtgtggtgtcgctcttgctatcctggtaggcactgcatcatcagcagcttgcatcgcca 9600 aagcaagaagagactgcctgacgccatacgcgcttgcaccgaacgcaacggtacccacag 9660 cattagcggttttgtgctgcattcggccaaccaacgctgaaacatttggagaaactttga 9720 accatctgtggtttaacaaccaaccgtttctctgggcacagttgtgcattcctctggcag 9780 cgcttgttattctgttccgctgcttttcatgctgcatgccttttttattggttgcaggcg 9840 tctgcctggggaaggtagacgccttcgaacatgcgaccactgtgccaaatgttccgggga 9900 tcccgtataaggcgttggtcgaacgcgcaggttacgcgccacttaacctggagatcacgg 9960 tcgtctcatcggaattaacaccttcaactaacaaggagtacgtgacctgcaaattccaca 10020 cagtcattccttcaccacaagttaaatgctgcgggtccctcgagtgcaaggcatcctcaa 10080 aggcggattacacatgccgcgtttttggcggtgtgtaccctttcatgtggggaggcgcac 10140 aatgcttctgtgacagtgagaacacacaactgagtgaggcgtacgtcgagttcgctccag 10200 actgcactatagatcacgcagtcgcactaaaagttcacacagctgctctgaaagtcggcc 10260 tgcgtatagtatacggcaacaccaccgcgcacctggatacgtttgtcaatggcgtcacgc 10320 caggttcctcacgggacctgaaggtcatagcagggccgatatcagccgctttttcaccct 10380 ttgaccataaggtcgtcatcagaaaggggcttgtttacaactacgacttccctgagtatg 10440 gagctatgaaaccaggagcgttcggcgatattcaagcatcctcgcttgatgctacagaca 10500 tagtagcccgcactgacatacggctgctgaagccttctgtcaagaacatccacgtcccct 10560 acacccaagcagtatcagggtatgaaatgtggaagaacaactcaggacgacccctgcaag 10620 aaacagcaccatttggatgtaaaattgaagtggagcctctgcgagcgtctaactgtgctt 10680 acgggcacatccctatctcgattgacatccctgatgcagcttttgtgagatcatcagaat 10740 caccaacaattttagaagttagctgcacagtagcagactgcatttattctgcagactttg 10800 gtggttctctaacattacagtacaaagctgacagggagggacattgtccagttcactccc 10860 actccacgacagctgttttgaaggaagcgaccacacatgtgactgccgtaggcagcataa 10920 cactacattttagcacatcgagcccacaagcaaattttatagtttcgctatgcggcaaga 10980 agtccacctgcaatgctgaatgtaaaccaccggccgaccacataattggagaaccacata 11040 aagtcgaccaagaattccaggcggcagtttccaaaacatcttggaactggctgcttgcac 11100 tgtttgggggagcatcatccctcattgttgtaggacttatagtgttggtctgcagctcta 11160 tgcttataaacacacgtagatgactgagcgcggacactgacatagcggtaaaactcgatg 11220 tacttccgaggaagcgtggtgcataatgccacgcgccgcttgacactaaaactcgatgta 11280 tttccgaggaagcacagtgcataatgctgtgcagtgtcacattaatcgtatatcacacta 11340 catattaacaacactatatcacttttatgagactcactatgggtctctaatatacactac 11400 acatattttacttaaaaacactatacacactttataaattcttttataatttttcttttg 11460 tttttattttgtttttaaaatttc 11484 (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4150 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:

Canadian Seq-1416-l5ver2 rev apr 15 2002 (A) ORGANISM: western equine encephalomyelitis virus -strain 71v-1658 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 160-3869 (D) OTHER INFORMATION: vector sequence 1 - 9 5' Sacl primer 9 - 20 3' end of NS4 gene 16 - 114 intragenic region 115 - 158 polyprotein (C-E3-E2-6K-E1) 159 - 3856 pcDw-xH7 nontranslated region 3857 - 4150 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
ggccctctagagctcatactggcaggcctgatcatcacgtctctgtccacgttagccgaa 60 agcgttaagaacttcaagagcataagagggagcccaatcaccctctacggctgacctaaa 120 taggtgacgtagtagacacgcacctacccaccggcagaatgtttccataccctcagctga 180 actttccaccagtttaccctacaaatccgatggcttaccgagatccaaaccctcctaggc 240 gccgctggaggccgtttcggcccccgctggctgctcaaatcgaagatcttaggaggtcga 300 tagtcaacttgactttcaaacaacgatcacctaatccgccgccaggtccaccgccaaaga 360 agaagaagagtgctcctaagccaaaacctactcagcctaaaaagaagaagcagcaagcca 420 agaggacgaaacgcaagcctaaaccagggaaacgacaacgtatgtgtatgaagttggagt 480 cggacaagacatttccgatcatgctgaacggccaagtgaatggatatgcctgcgttgtcg 540 gaggaaggctgatgaaaccactccacgttgaaggaaaaattgataatgagcaattagcgg 600 ccgtgaaattgaagaaggctagcatgtacgacttggagtacggcgacgttccccagaaca 660 tgaaatcagacacgctgcagtacaccagcgacaaaccaccgggcttctacaactggcacc 720 acggcgcagtccagtatgagaatgggagatttaccgtaccgagaggagtgggcgggaaag 780 gcgacagcggaagaccgatcctggacaacagaggcagagttgtggctattgttctaggag 840 gtgcaaatgagggcacgcgtacggcgctttcagtggtcacttggaaccagaaaggggtga 900 ccattagggatacccccgaaggttctgaaccgtggtcactagttacagcgctatgcgtgc 960 tttcgaatgtcacgttcccatgcgacaaaccacccgtgtgctattcactgacgccagaac 1020 gaacactcgacgtgctcgaagagaacgtcgacaatccaaattacgacacgctgctggaga 1080 acgtcttgaaatgtccatcacgccggcccaaacgaagcattaccgatgacttcacactga 1140 ccagtccctacctggggttctgcccgtattgcagacactcaacgccgtgtttcagcccaa 1200 taaaaattgagaacgtgtgggacgaatctgatgatggatcgattagaatccaggtctcgg 1260 cacaattcggctacaatcaggcaggcactgcggatgtcaccaaattccgttacatgtctt 1320 tcgaccacgaccatgacatcaaggaagacagtatggagaaaatagctatcagcacatctg 1380 gaccctgccgtcgtcttggccacaaagggtacttcctgttagctcaatgtcctccaggtg 1440 acagtgtaaccgtcagtatcacgagcggagcatctgagaattcatgcaccgtggagaaaa 1500 agatcaggaggaagtttgtcggtagagaggagtacttgttcccacccgtccatggaaagc 1560 tggtaaagtgccacgtttacgatcacttgaaggagacgtctgccgggtacataaccatgc 1620 acaggccaggcccacacgcgtataagtcctatctggaggaagcgtcaggcgaagtgtaca 1680 ttaaaccaccttctggcaagaacgtcacctacgaatgtaagtgtggcgactacagcacag 1740 gtatcgtgagcacgcgaacgaagatgaacggctgcactaaagcaaaacagtgcattgcct 1800 acaagagcgaccaaacgaaatgggtcttcaactcgccggatcttattaggcacacagacc 1860 actcagtgcaaggtaaattgcacattccattccgcttgacaccgacagtctgcccggttc 1920 cgttagctcacacgcctacagtcacgaagtggttcaaaggcatcaccctccacctgactg 1980 caatgcgaccaacattgctgacaacgagaaaattggggctgcgagcagacgcaacagcag 2040 aatggattacagggtctacatccaggaatttttctgtggggcgagaagggctggagtacg 2100 tatggggtaaccatgaaccagtcagagtctgggcccaggagtcggcaccaggcgacccac 2160 atggatggccgcatgagatcatcatccactattatcatcggcatccagtctacactgtca 2220 ttgtgctgtgtggtgtcgctcttgctatcctggtaggcactgcatcatcagcagcttgca 2280 tcgccaaagcaagaagagactgcctgacgccatacgcgcttgcaccgaacgcaacggtac 2340 ccacagcattagcggttttgtgctgcattcggccaaccaacgctgaaacatttggagaaa 2400 ctttgaaccatctgtggtttaacaaccaaccgtttctctgggcacagttgtgcattcctc 2460 tggcagcgcttgttattctgttccgctgcttttcatgctgcatgccttttttattggttg 2520 caggcgtctgcctggggaaggtagacgccttcgaacatgcgaccactgtgccaaatgttc 2580 cggggatcccgtataaggcgttggtcgaacgcgcaggttacgcgccacttaacctggaga 2640 tcacggtcgtctcatcggaattaacaccttcaactaacaaggagtacgtgacctgcaaat 2700 tccacacagtcattccttcaccacaagttaaatgctgcgggtccctcgagtgcaaggcat 2760 cctcaaaggcggattacacatgccgcgtttttggcggtgtgtaccctttcatgtggggag 2820 gcgcacaatgcttctgtgacagtgagaacacacaactgagtgaggcgtacgtcgagttcg 2880 ctccagactgcactatagatcacgcagtcgcactaaaagttcacacagctgctctgaaag 2940 tcggcctgcgtatagtatacggcaacaccaccgcgcacctggatacgtttgtcaatggcg 3000 tcacgccaggttcctcacgggacctgaaggtcatagcagggccgatatcagccgcttttt 3060 caccctttgaccataaggtcgtcatcagaaaggggcttgtttacaactacgacttccctg 3120 agtatggagctatgaaaccaggagcgttcggcgatattcaagcatcctcgcttgatgcta 3180 Canadian -1416-l5ver2 Seq rev apr cagacatagtagcccgcactgacatacggctgctgaagccttctgtcaagaacatccacg 3240 tcccctacacccaagcagtatcagggtatgaaatgtggaagaacaactcaggacgacccc 3300 tgcaagaaacagcaccatttggatgtaaaattgaagtggagcctctgcgagcgtctaact 3360 gtgcttacgggcacatccctatctcgattgacatccctgatgcagcttttgtgagatcat 3420 cagaatcaccaacaattttagaagttagctgcacagtagcagactgcatttattctgcag 3480 actttggtggttctctaacattacagtacaaagctgacagggagggacattgtccagttc 3540 actcccactccacgacagctgttttgaaggaagcgaccacacatgtgactgccgtaggca 3600 gcataacactacattttagcacatcgagcccacaagcaaattttatagtttcgctatgcg 3660 gcaagaagtccacctgcaatgctgaatgtaaaccaccggccgaccacataattggagaac 3720 cacataaagtcgaccaagaattccaggcggcagtttccaaaacatcttggaactggctgc 3780 ttgcactgtttgggggagcatcatccctcattgttgtaggacttatagtgttggtctgca 3840 gctctatgcttataaacacacgtagatgactgagcgcggacactgacatagcggtaaaac 3900 tcgatgtacttccgaggaagcgtggtgcataatgccacgcgccgcttgacactaaaactc 3960 gatgtatttccgaggaagcacagtgcataatgctgtgcagtgtcacattaatcgtatatc 4020 acactacatattaacaacactatatcacttttatgagactcactatgggtctctaatata 4080 cactacacatattttacttaaaaacactatacacactttataaattctctcataatttca 4140 ctttaggttt 4150 (2) INFORMATION FOR SEQ ID NO: 3:
(i)SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4395 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA

(vi)ORIGINAL SOURCE:

(A) ORGANISM: western equine encephalomyelitis virus -strain 71V-1658 (vii) IMMEDIATE SOURCE: Example of a CMV coupled the promoter to pcdw-xh7 sequence (ix)FEATURE:

(A) NAME/KEY: CMV promoter (B) LOCATION: 1 .. 1260 (D) OTHER INFORMATION: pvAX vector 1 - 196 sequence CMV promoter 1 - 11 5 CMV putative transcriptional start site 125 T7 promoter 148 - 167 PvAx mulitcloning region 168 - 196 polyprotein (C-E3-E2-6K-E1) 214 - 4065 pcDw-Hx45 nontranslated region 4066 - 4348 pcDw-Hx45 vector sequence 4349 - 4385 pvAx vector sequence 4386 (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 3:
accaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgg 60 gcggtaggcgtgtacggtgggaggtcatatataagcagagtctctctggctaactagaga 120 acccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagctgg 180 ctagcgtttaaacttaagcttggtaccgagctcatactggcaggcctgatcatcacgtct 240 ctgtccacgttagccgaaagcgttaagaacttcaagagcataagagggagcccaatcacc 300 ctctacggctgacctaaataggtgacgtagtagacacgcacctacccaccgccagaatgt 360 ttccataccctcagctgaactttccaccagtttaccctacaaatccgatggcttaccgag 420 atccaaaccctcctaggcgccgctggaggccgtttcggcccccgctggctgctcaaatcg 480 aagatcttaggaggtcgatagtcaacttgactttcaaacaacgatcacctaatccgccgc 540 caggtccaccgccaaagaagaagaagagtgctcctaagccaaaacctactcagcctaaaa 600 agaagaagcagcaagccaagaggacgaaacgcaagcctaaaccagggaaacgacaacgta 660 tgtgtatgaagttggagtcggacaagacatttccgatcatgctgaacggccaagtgaatg 720 gatatgcctgcgttgtcggaggaaggctgatgaaaccactccacgttgaaggaaaaattg 780 ataatgagcaattagcggccgtgaaattgaagaaggctagcatgtacgacttggagtacg 840 gcgacgttccccagaacatgaaatcagacacgctgcagtacaccagcgacaaaccaccgg 900 gcttctacaactggcaccacggcgcagtccagtatgagaatgggagatttaccgtaccga 960 Canadian -1416-l5ver2 Seq rev apr gaggagtgggcgggaaaggcgacagcggaagaccgatcctggacaacagaggcagagttg 1020 tggctattgttctaggaggtgcaaatgagggcacgcgtacggcgctttcagtggtcactt 1080 ggaaccagaaaggggtgaccattagggatacccccgaaggttctgaaccgtggtcactag 1140 ttacagcgctatgcgtgctttcgaatgtcacgttcccatgcgacaaaccacccgtgtgct 1200 attcactgacgccagaacgaacactcgacgtgctcgaagagaacgtcgacaatccaaatt 1260 acgacacgctgctggagaacgtcttgaaatgtccatcacgccggcccaaacgaagcatta 1320 ccgatgacttcacactgaccagtccctacctggggttctgcccgtattgcagacactcaa 1380 cgccgtgtttcagcccaataaaaattgagaacgtgtgggacgaatctgatgatggatcga 1440 ttagaatccaggtctcggcacaattcggctacaatcaggcaggcactgcggatgtcacca 1500 aattccgttacatgtctttcgaccacgaccatgacatcaaggaagacagtatggagaaaa 1560 tagctatcagcacatctggaccctgccgtcgtcttggccacaaagggtacttcctgttag 1620 ctcaatgtcctccaggtgacagtgtaaccgtcagtatcacgagcggagcatctgagaatt 1680 catgcaccgtggagaaaaagatcaggaggaagtttgtcggtagagaggagtacttgttcc 1740 cacccgtccatggaaagctggtaaagtgccacgtttacgatcacttgaaggagacgtctg 1800 ccgggtacataaccatgcacaggccaggcccacacgcgtataagtcctatctggaggaag 1860 cgtcaggcgaagtgtacattaaaccaccttctggcaagaacgtcacctacgaatgtaagt 1920 gtggcgactacagcacaggtatcgtgagcacgcgaacgaagatgaacggctgcactaaag 1980 caaaacagtgcattgcctacaagagcgaccaaacgaaatgggtcttcaactcgccggatc 2040 ttattaggcacacagaccactcagtgcaaggtaaattgcacattccattccgcttgacac 2100 cgacagtctgcccggttccgttagctcacacgcctacagtcacgaagtggttcaaaggca 2160 tcaccctccacctgactgcaatgcgaccaacattgctgacaacgagaaaattggggctgc 2220 gagcagacgcaacagcagaatggattacagggtctacatccaggaatttttctgtggggc 2280 gagaagggctggagtacgtatggggtaaccatgaaccagtcagagtctgggcccaggagt 2340 cggcaccaggcgacccacatggatggccgcatgagatcatcatccactattatcatcggc 2400 atccagtctacactgtcattgtgctgtgtggtgtcgctcttgctatcctggtaggcactg 2460 catcatcagcagcttgcatcgccaaagcaagaagagactgcctgacgccatacgcgcttg 2520 caccgaacgcaacggtacccacagcattagcggttttgtgctgcattcggccaaccaacg 2580 ctgaaacatttggagaaactttgaaccatctgtggtttaacaaccaaccgtttctctggg 2640 cacagttgtgcattcctctggcagcgcttgttattctgttccgctgcttttcatgctgca 2700 tgccttttttattggttgcaggcgtctgcctggggaaggtagacgccttcgaacatgcga 2760 ccactgtgccaaatgttccggggatcccgtataaggcgttggtcgaacgcgcaggttacg 2820 cgccacttaacctggagatcacggtcgtctcatcggaattaacaccttcaactaacaagg 2880 agtacgtgacctgcaaattccacacagtcattccttcaccacaagttaaatgctgcgggt 2940 ccctcgagtgcaaggcatcctcaaaggcggattacacatgccgcgtttttggcggtgtgt 3000 accctttcatgtggggaggcgcacaatgcttctgtgacagtgagaacacacaactgagtg 3060 aggcgtacgtcgagttcgctccagactgcactatagatcacgcagtcgcactaaaagttc 3120 acacagctgctctgaaagtcggcctgcgtatagtatacggcaacaccaccgcgcacctgg 3180 atacgtttgtcaatggcgtcacgccaggttcctcacgggacctgaaggtcatagcagggc 3240 cgatatcagccgctttttcaccctttgaccataaggtcgtcatcagaaaggggcttgttt 3300 acaactacgacttccctgagtatggagctatgaaaccaggagcgttcggcgatattcaag 3360 catcctcgcttgatgctacagacatagtagcccgcactgacatacggctgctgaagcctt 3420 ctgtcaagaacatccacgtcccctacacccaagcagtatcagggtatgaaatgtggaaga 3480 acaactcaggacgacccctgcaagaaacagcaccatttggatgtaaaattgaagtggagc 3540 ctctgcgagcgtctaactgtgcttacgggcacatccctatctcgattgacatccctgatg 3600 cagcttttgtgagatcatcagaatcaccaacaattttagaagttagctgcacagtagcag 3660 actgcatttattctgcagactttggtggttctctaacattacagtacaaagctgacaggg 3720 agggacattgtccagttcactcccactccacgacagctgttttgaaggaagcgaccacac 3780 atgtgactgccgtaggcagcataacactacattttagcacatcgagcccacaagcaaatt 3840 ttatagtttcgctatgcggcaagaagtccacctgcaatgctgaatgtaaaccaccggccg 3900 accacataattggagaaccacataaagtcgaccaagaattccaggcggcagtttccaaaa 3960 catcttggaactggctgcttgcactgtttgggggagcatcatccctcattgttgtaggac 4020 ttatagtgttggtctgcagctctatgcttataaacacacgtagatgactgagcgcggaca 4080 ctgacatagcggtaaaactcgatgtacttccgaggaagcgtggtgcataatgccacgcgc 4140 cgcttgacactaaaactcgatgtatttccgaggaagcacagtgcataatgctgtgcagtg 4200 tcacattaatcgtatatcacactacatattaacaacactatatcacttttatgagactca 4260 ctatgggtctctaatatacactacacatattttacttaaaaacactatacacactttata 4320 aattcttttataatttttcttttgctttagagcacactggcggccgttactagtggatcc 4380 gagctctagagggcc 4395

Claims (17)

1. A western equine encephalitis ("WEE") virus strain 71V-1658 comprising the nucleotide sequence shown in SEQ ID NO: 1.

2. A process for preparing a recombinant DNA vaccine for inducing protective immune response to WEE virus in a mammal, comprising preparing a nucleic acid suitable for producing antigenic determinant in a mammal in vivo by encoding antigenic determinant of WEE virus strain 71V-1658 structural proteins operatively linked to a mammalian expression promoter.

3. A process for preparing a recombinant DNA vaccine according to claim 2, wherein said mammalian expression promoter is a cytomegalovirus promoter.

4. A process for preparing a recombinant DNA vaccine according to claim 2 or 3, wherein said structural proteins are selected from the group consisting of capsid, E1 protein, E2 protein, E3 protein and the 26S polyprotein gene segment of WEE
virus strain 71V-1658.

5. A process for preparing a recombinant DNA vaccine according to claim 2, 3 or 4, wherein said nucleic acid is naked.

6. A process for preparing a recombinant DNA vaccine according to claim 2,3, or 4, wherein said nucleic acid is encapsulated in liposomes.

7. A process for preparing a recombinant DNA vaccine according to claim 2, 3 or 4, wherein said nucleic acid is coated onto gold particles.

8. A prophylactic method for inducing protective immune response to WEE virus in a mammal comprising:
(i) preparing a nucleic acid suitable for producing antigenic determinant in a mammal in vivo by encoding antigenic determinant of WEE virus strain 71V-1658 structural proteins operatively linked to a mammalian expression promoter; and (ii) delivering said nucleic; acid into the mammal.

9. A prophylactic method for inducing protective immune response to WEE virus in a mammal according to claim 8, wherein said mammalian expression promoter is a cytomegalovirus promoter.

10. A prophylactic method for inducing protective immune response to WEE virus in a mammal according to claim 8 or 9, wherein said delivery is effected via an intramusular injection.

11. A prophylactic method for inducing protective immune response to WEE virus in a mammal according to claim 8 or 9, wherein said delivery is effected via an aerosol spray.

12. A prophylactic method for inducing protective immune response to WEE virus in a mammal according to claim 8 or 9, wherein said delivery is effected via an accerating gold particles coated with said nuclei acid.

13. A prophylactic method for inducing protective immune response to WEE virus in a mammal according to claim 8 or 9, wherein said delivery of said nucleic acid is via liposomal encapsulation.

14. A prophylactic method according to any of claim 8, 9, 10, 11, 12 or 13, for inducing a protective immune response to eastern equine encephalitis virus and Venezuelan equine encephalitis virus in a mammal.

15. A structural gene pcDWXH-7 comprising nucleotide sequence shown in SEQ ID
NO:
2.

16. A recombinant DNA expression vector pVHX-6 comprising upstream nucleotide sequence shown in SEQ ID NO: 3 and having remaining nucleotide sequence identical to that of structural gene pcDWXH-7 of SEQ ID NO: 2 from the point of divergence.

17. A recombinant DNA vaccine for inducing protective immune response to WEE
virus, wherein structural proteins of WEE virus SEQ ID NO:2 are operationally linked to a cytomegalovirus promoter in a nucleic acid pVHX-6 of SEQ ID NO: 3.