CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Serial No. 61/620,122, filed 4 April 2012, which is herein incorporated by reference in its entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The content of the ASCII text file of the sequence listing named

"20120523_034047_057_seq_ST25" which is 54.6 kb in size was created on 23 May 2012 and electronically submitted via EFS-Web herewith the application is incorporated herein by reference in its entirety.

ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

This invention was made by employees of the United States Army Medical Research and Materiel Command, which is an agency of the United States

Government. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. FIELD OF THE INVENTION.

The present invention generally relates to nucleic acid molecules derived from equine encephalitis viruses and compositions and methods thereof.

2. DESCRIPTION OF THE RELATED ART.

Venezuelan equine encephalitis virus (VEEV), eastern equine encephalitis virus (EEEV), and western equine encephalitis virus (WEEV) are non-segmented, positive-sense RNA viruses of the genus Alphavirus in the family Togaviridae. See Griffin (2001) "Alphaviruses", p. 917-962, in FIELDS VIROLOGY, vol. 4. Lippincott, Williams, and Wilkins, Philadelphia, PA. Naturally transmitted by mosquitoes through rodent or bird hosts, VEEV, EEEV, and WEEV are highly pathogenic for equines and humans and have caused periodic epizootics throughout North, Central, and South America. See Tsai (1991) Infect Dis Clin North Am 5:73-102. Human infection with these New World alphaviruses typically results in an acute,

incapacitating disease characterized by fever, headache, lymphopenia, myalgia, and malaise. See Bale (1993) Med Clin North Am 77:25-42. Severe neurological disease, including fatal encephalitis, can also result from VEEV, EEEV, and WEEV infection of humans. Although the human case-fatality rates are estimated to be low for VEEV (<1%) and WEEV (8-15%), EEEV is the most severe of the arbovirus encephalitides with a human case-fatality rate estimated to be 30-70%. See Steele et al. (2007) "Alphavirus Encephalitides" p. 241-270, in MEDICAL ASPECTS OF BIOLOGICAL WARFARE. BORDEN INSTITUTE (U.S. Army Walter Reed), Washington, DC.

However, numerous documented laboratory accidents and the results of animal studies have demonstrated that VEEV, EEEV, and WEEV are also highly infectious in aerosols, and infection with aerosolized virus could potentially result in higher mortality than that observed with natural infection. See Franz et al. (2001) Clin Lab Med 21 :435-73; Hanson (1967) Science 158: 1283-6; and Kortepeter et al. (2001) J Environ Health 63:21-4. In addition to producing incapacitating or lethal infections and being infectious in aerosols, these encephalitic alphaviruses are also easily grown to high titers in inexpensive and unsophisticated cell culture systems and are relatively stable. As a result, VEEV, EEEV, and WEEV represent significant potential biological defense threats and are classified as Category B priority biodefense agents by both the Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases.

[12] Although there are no licensed human vaccines for the encephalitic

alphaviruses, live-attenuated and formalin-inactivated vaccines are currently being utilized under Investigational New Drug (IND) status to protect laboratory workers and other at-risk personnel. A live-attenuated vaccine for VEEV, TC-83, provides long-lasting immunity and protection from both subcutaneous and aerosol VEEV challenges; however, it causes adverse reactions in approximately 25%> of recipients, and approximately 20% of recipients fail to develop a detectable neutralizing antibody response. See McKinney et al. (1963) Am J Trop Med Hyg 12:597-603; and Pittman et al. (1996) Vaccine 14:337-43. C-84 (formalin-inactivated TC-83 VEEV vaccine), and EEEV and WEEV formalin-inactivated vaccines are well tolerated, but they require frequent boosting to elicit detectable neutralizing antibody responses in humans and have provided poor protection against aerosol viral challenge in animal studies. See Cole et al. (1973) Appl Microbiol 25:262-5; Bartelloni et al. (1970) Am J Trop Med Hyg 19:123-6; and Bartelloni et al. (1971) Am J Trop Med Hyg 20: 146-9. Due to the significant limitations associated with the existing live-attenuated and formalin-inactivated vaccines currently being utilized under IND status, the development of improved vaccines that can safely and effectively protect against encephalitic alphavirus infections in humans is needed. [13] Next-generation VEEV vaccines, including live-attenuated, inactivated, attenuated Sindbis/VEEV chimeric viruses, alphavirus replicons, and DNA vaccines, are all currently at various stages of development. See Paessler & Weaver (2009) Vaccine 27 Suppl 4:D80-5. Genetic vaccination with DNA plasmids expressing immunogenic proteins has numerous inherent advantages as a platform for the development of next-generation vaccines. Among the benefits of this method are that DNA vaccines can be rapidly and cost-effectively produced without the need to propagate a pathogen, do not require the inactivation of infectious organisms, avoid problems of preexisting or vector-induced immunity due to lack of a host immune response to the plasmid backbone, and have exhibited a favorable safety profile in numerous human clinical trials. See Dupuy & Schmaljohn (2009) Expert Rev Vaccines 8:1739-54.

[14] In previous studies, mice vaccinated with a DNA vaccine expressing the

structural proteins (C-E3-E2-6K-E1) of VEEV subtype IAB (strain Trinidad Donkey) by particle-mediated epidermal delivery (PMED) or "gene gun", in which plasmid DNA-coated gold particles are delivered intradermally in a ballistic manner, developed strong overall antibody responses against VEEV IAB. Unfortunately, the VEEV-neutralizing antibody responses were low, and only 80% protection against lethal aerosol challenge was observed. See Riemenschneider et al. (2003) Vaccine 21 :4071 -80. Cynomolgus macaques vaccinated with this VEEV DNA vaccine by PMED developed detectable levels of VEEV IAB -neutralizing antibodies, but only partial protection was observed upon aerosol challenge. See Dupuy et al. (2010) Vaccine 28:7345-50.

[15] In other studies to develop a human vaccine for encephalitic alphaviruses, directed molecular evolution or "gene shuffling" of the envelope protein genes was used as an attempt to improve the neutralizing antibody response to VEEV, EEEV, and WEEV DNA vaccines. DNA vaccines expressing representative variants from a library in which the E2 envelope glycoprotein genes of five parent viruses (VEEV subtypes IAB and IE, Mucambo virus, EEEV (strain PE6), and WEEV (strain CBA87) were recombined and the El envelope glycoprotein gene of VEEV IAB was held constant elicited significantly increased neutralizing antibody titers to VEEV IAB compared to the wild-type parent VEEV DNA vaccine and provided improved protection against aerosol VEEV IAB challenge in mice. See Dupuy et al. (2009) Vaccine 27:4152-60. Unfortunately, in addition to the in vitro gene recombination being technically difficult and the screening of variants for improved immunogenicity being labor-intensive, the studies failed to result in variant envelope glycoprotein vaccines having improved immunogenicity against EEEV and WEEV as compared to the wild-type parent EEEV and WEEV DNA vaccines.

[16] Therefore, a need still exists for safe and effective vaccines to protect against equine encephalitis viruses (EEVs) such as VEEV, EEEV, and WEEV.

[ 17] SUMMARY OF THE INVENTION

[18] The present invention provides nucleotide sequence which encodes a plurality of structural proteins, except the capsid, of an equine encephalitis virus, wherein the nucleotide sequence is codon-optimized for mammalian expression. In some embodiments, the nucleotide sequences are codon-optimized for expression in humans. In some embodiments, the structural proteins of the plurality of structural proteins are selected from the group consisting of E3, E2, 6K and El . In some embodiments, the plurality of structural proteins comprises, consists essentially of, or consists of E3, E2, 6K and El . In some embodiments, the equine encephalitis virus is Venezuelan equine encephalitis virus (VEEV), western equine encephalitis virus (WEEV), or eastern equine encephalitis virus (EEEV). In some embodiments, the vector sequence is that of eukaryotic expression vector pWRG7077. In some embodiments, the nucleotide sequence has at least about 85%, preferably about 90%, more preferably about 95%, or most preferably about 99% or more sequence identity to SEQ ID NO: l, SEQ ID NO:3, or SEQ ID NO:5. In some embodiments, the nucleotide sequence is selected from the group consisting of SEQ ID NO: l, SEQ ID NO:3, and SEQ ID NO:5. In some embodiments, the nucleotide sequence are provided in the form of a plasmid, i.e. are contained within a vector sequence, such as the eukaryotic expression vector pWRG7077.

[19] In some embodiments, the present invention provides compositions

comprising, consisting essentially of, or consisting of one or more EEVco

polynucleotides and/or one or more EEVco antibodies raised against the one or more EEVco polynucleotides. Such EEVco polynucleotides comprise, consist essentially of, or consist of a nucleotide sequence which encodes a plurality of structural proteins, except the capsid, of an equine encephalitis virus, wherein the nucleotide sequence is codon-optimized for mammalian expression. In some embodiments, the nucleotide sequences are codon-optimized for expression in humans. In some embodiments, the structural proteins of the plurality of structural proteins are selected from the group consisting of E3, E2, 6K and El . In some embodiments, the plurality of structural proteins comprises, consists essentially of, or consists of E3, E2, 6K and El . In some embodiments, the equine encephalitis virus is Venezuelan equine encephalitis virus (VEEV), western equine encephalitis virus (WEEV), or eastern equine encephalitis virus (EEEV). In some embodiments, the EEVco polynucleotides are contained within a vector sequence such as the eukaryotic expression vector pWRG7077. In some embodiments, the nucleotide sequence has at least about 85%, preferably about 90%, more preferably about 95%, or most preferably about 99% or more sequence identity to SEQ ID NO: l , SEQ ID NO: 3, or SEQ ID NO:5. In some embodiments, the nucleotide sequence is selected from the group consisting of SEQ ID NO: l , SEQ ID NO:3, and SEQ ID NO:5. In some embodiments, the EEV _C0 polynucleotide is provided in the form of an EEVco plasmid, i.e. contained within a vector sequence such as the eukaryotic expression vector pWRG7077. In some embodiments, the sequence of the EEVco plasmid has at least about 85%>, preferably about 90%), more preferably about 95%>, or most preferably about 99%> or more sequence identity to SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. In some embodiments, the compositions of the present invention further comprise

pharmaceutically acceptable carriers and/or adjuvants. In some embodiments, the one or more EEVco polynucleotides are provided in the compositions in immunogenic amounts and/or therapeutically effective amounts. In some embodiments, the one or more EEVco antibodies are provided in the compositions in therapeutically effective amounts.

[20] In some embodiments, the present invention provides methods of eliciting an immune response in a subject, preferably a mammalian subject, more preferably a primate, most preferably a human, which comprises, consists essentially of, or consists of administering to the subject at least one immunogenic amount of (a) at least one EEVco polynucleotide or at least one EEVco plasmid as disclosed herein, (b) at least one plasmid comprising, consisting essentially of, or consisting of a vector sequence and a nucleotide sequence that has at least about 85%>, preferably about 90%o, more preferably about 95%>, or most preferably about 99%> or more sequence identity to SEQ ID NO: l , SEQ ID NO:3, or SEQ ID NO:5; (c) at least one plasmid comprising, consisting essentially of, or consisting of a vector sequence and a nucleotide sequence selected from the group consisting of SEQ ID NO: l , SEQ ID NO:3, and SEQ ID NO:5; (d) a composition comprising, consisting essentially of, or consisting of one or more plasmids according to (a)-(c) above; or (e) a composition comprising, consisting essentially of, or consisting of nucleotide sequences having SEQ ID NO: l, SEQ ID NO:3, and SEQ ID NO:5, wherein the nucleotide sequences may be contained within one or more vector sequences. In some embodiments, the EEVco polynucleotides comprise, consist essentially of, or consist of a nucleotide sequence which encodes a plurality of structural proteins, except the capsid, of an equine encephalitis virus, wherein the nucleotide sequence is codon-optimized for mammalian expression. In some embodiments, the nucleotide sequences are codon- optimized for expression in humans. In some embodiments, the structural proteins of the plurality of structural proteins are selected from the group consisting of E3, E2, 6K and El . In some embodiments, the plurality of structural proteins comprises, consists essentially of, or consists of E3, E2, 6K and El . In some embodiments, the equine encephalitis virus is Venezuelan equine encephalitis virus (VEEV), western equine encephalitis virus (WEEV), or eastern equine encephalitis virus (EEEV). In some embodiments the EEVco polynucleotides are contained within a vector sequence such as the eukaryotic expression vector pWRG7077. In some

embodiments, the immune response is a cellular immune response. In some embodiments, the immune response is one that is observable and/or measurable using methods known in the art. In some embodiments, the amount of the immune response is a total IgG antibody response and/or a neutralizing antibody response that is significantly more than that provided by a corresponding EEVW _T control. In some embodiments, the amount of the immune response is a total IgG antibody response and/or a neutralizing antibody response that is substantially similar to that provided by TC-83. In some embodiments, the immunogenic amount is one that results in an immunogenic response as compared to a negative control. In some embodiments, the immunogenic amount is about 10-1250 μg/kg subject. In some embodiments, the immunogenic amount is administered as separate doses at different times. In some embodiments, an additional immunogenic amount, e.g. booster dose, is subsequently administered. In some embodiments, the immunogenic amounts are administered by a particle-mediated epidermal delivery method.

[21] In some embodiments, the present invention provides methods of immunizing a subject, preferably a mammalian subject, more preferably a primate, most preferably a human, against one or more equine encephalitis viruses which comprises, consists essentially of, or consists of administering to the subject at least one immunogenic amount of (a) at least one EEVco polynucleotide or at least one EEVco plasmid as disclosed herein, (b) at least one plasmid comprising, consisting essentially of, or consisting of a vector sequence and a nucleotide sequence that has at least about 85%, preferably about 90%, more preferably about 95%, or most preferably about 99% or more sequence identity to SEQ ID NO: l, SEQ ID NO:3, or SEQ ID NO:5; (c) at least one plasmid comprising, consisting essentially of, or consisting of a vector sequence and a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO:3, and SEQ ID NO:5; (d) a composition comprising, consisting essentially of, or consisting of one or more plasmids according to (a)-(c) above; or (e) a composition comprising, consisting essentially of, or consisting of nucleotide sequences having SEQ ID NO: l, SEQ ID NO:3, and SEQ ID NO:5, wherein the nucleotide sequences may be contained within one or more vector sequences. In some embodiments, the EEVco polynucleotides comprise, consist essentially of, or consist of a nucleotide sequence which encodes a plurality of structural proteins, except the capsid, of an equine encephalitis virus, wherein the nucleotide sequence is codon-optimized for mammalian expression. In some embodiments, the nucleotide sequences are codon- optimized for expression in humans. In some embodiments, the structural proteins of the plurality of structural proteins are selected from the group consisting of E3, E2, 6K and El . In some embodiments, the plurality of structural proteins comprises, consists essentially of, or consists of E3, E2, 6K and El . In some embodiments, the equine encephalitis virus is Venezuelan equine encephalitis virus (VEEV), western equine encephalitis virus (WEEV), or eastern equine encephalitis virus (EEEV). In some embodiments the EEVco polynucleotides are contained within a vector sequence such as the eukaryotic expression vector pWRG7077. In some

embodiments, the immune response is a cellular immune response. In some embodiments, the immune response is one that is observable and/or measurable using methods known in the art. In some embodiments, the amount of the immune response is a total IgG antibody response and/or a neutralizing antibody response that is significantly more than that provided by a corresponding EEVW _T control. In some embodiments, the amount of the immune response is a total IgG antibody response and/or a neutralizing antibody response that is substantially similar to that provided by TC-83. In some embodiments, the immunogenic amount is one that results in an immunogenic response as compared to a negative control. In some embodiments, the immunogenic amount is about 10-1250 μg/kg subject. In some embodiments, the immunogenic amount is administered as separate doses at different times. In some embodiments, an additional immunogenic amount, e.g. booster dose, is subsequently administered. In some embodiments, the immunogenic amounts are administered by a particle-mediated epidermal delivery method. In some embodiments, the immunogenic amount confers to the subject 100% survivability against exposure to one or more equine encephalitis viruses selected from the group consisting of Venezuelan equine encephalitis virus (VEEV), western equine encephalitis virus (WEEV), or eastern equine encephalitis virus (EEEV). In some embodiments, the one or more equine encephalitis viruses are aerosolized. In some embodiments, the immunogenic amount is administered by a particle-mediated epidermal delivery method.

[22] In some embodiments, the present invention provides a vaccine which

comprises, consists essentially of, or consists of an immunogenic amount of polynucleotides having SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. In some embodiments, the vaccine may further comprise one or more pharmaceutically acceptable carriers and/or adjuvants. In some embodiments, when administered to a mammalian subject, preferably a primate, more preferably a human, the vaccine confers to the subject 100% survivability against exposure to one or more equine encephalitis viruses selected from the group consisting of Venezuelan equine encephalitis virus (VEEV), western equine encephalitis virus (WEEV), or eastern equine encephalitis virus (EEEV). In some embodiments, the one or more equine encephalitis viruses are aerosolized. In some embodiments, the vaccine is administered by a particle-mediated epidermal delivery method.

[23] In some embodiments, the present invention provides kits which comprise one or more EEVco polynucleotides, one or more EEVco plasmids, one or more EEVco antibodies, and/or compositions thereof packaged together with a delivery device for administration to a subject. The kits may further include instructions for use. The one or more EEVco polynucleotides, one or more EEVco plasmids, one or more EEVco antibodies, and/or compositions may be provided in single unit dosage forms and the kits may comprise one or more single unit dosages.

[24] In some embodiments, the present invention provides one or more EEVco polynucleotides, one or more EEVco plasmids, one or more EEVco antibodies, and/or compositions thereof for use as a medicament. In some embodiments, the present invention provides use of one or more EEVco polynucleotides, one or more EEVco plasmids, one or more EEVco antibodies, and/or compositions thereof for the immunization of a subject. In some embodiments, the present invention provides one or more EEVco polynucleotides, one or more EEVco plasmids, one or more EEVco antibodies, and/or compositions thereof for the immunization of a subject. In some embodiments, the present invention provides one or more EEVco polynucleotides, one or more EEVco plasmids, one or more EEVco antibodies, and/or compositions thereof for use in immunizing a subject against infection by an EEV, wherein the one or more EEVco polynucleotides, one or more EEVco plasmids, one or more EEVco antibodies, and/or compositions thereof are administered by a dosage regime which results in an immune response in the subject.

[25] In some embodiments, the present invention is directed to use of one or more

EEVco polynucleotides, one or more EEVco plasmids, one or more EEVco antibodies, and/or compositions thereof. In some embodiments, the present invention is directed to use of SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, and/or SEQ ID NO:9. In some embodiments, the present invention is directed to use of one or more EEVco polynucleotides, one or more EEVco plasmids, one or more EEVco antibodies, and/or compositions thereof for eliciting an immune response in a subject and/or immunizing a subject against infection by an EEV. In some embodiments, the present invention is directed to use of SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, and/or SEQ ID NO:9 for eliciting an immune response in a subject and/or immunizing a subject against infection by an EEV. In some embodiments, the present invention is directed to use of one or more EEVco polynucleotides, one or more EEVco plasmids, one or more EEVco antibodies, and/or compositions thereof for the manufacture of a medicament for eliciting an immune response in a subject and/or immunizing a subject against infection by an EEV, wherein the medicament is administered in an immunogenic amount and/or by a particle-mediated epidermal delivery method. In some embodiments, the present invention is directed to use of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, and/or SEQ ID NO:9 for the manufacture of a medicament for eliciting an immune response in a subject and/or immunizing a subject against infection by an EEV, wherein the medicament is administered in an immunogenic amount and/or by a particle-mediated epidermal delivery method. In some embodiments, the present invention provides one or more EEVco polynucleotides, one or more EEVco plasmids, one or more EEVco antibodies, and/or compositions thereof for use in immunizing against an EEV, wherein the one or more EEVco polynucleotides, the one or more EEVco plasmids, the one or more EEVco antibodies, and/or the compositions thereof is/are

administered in an immunogenic amount and/or by a particle-mediated epidermal delivery method.

[26] Both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed. The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute part of this specification, illustrate several embodiments of the invention, and together with the description serve to explain the principles of the invention.

[27] DESCRIPTION OF THE DRAWINGS

[28] This invention is further understood by reference to the drawings wherein:

[29] Figs. 1A and IB are graphs summarizing the in vitro expression analysis in transfected cells. Radiolabeled lysates of COS-7 cells transiently trans fected with 5 μg of empty vector, VEEVW _T , or VEEVco DNA were immune precipitated with VEEV IAB hyperimmune mouse ascitic fluids (HMAF) and analyzed by SDS-PAGE and phosphor imaging (A). COS-7 cells transiently transfected with 5-250 ng of VEEVWT, VEEVCO, or VEEVCOCAP DNA were permeabilized, stained with a VEEV E2- or El -specific mouse mAb and a AlexaFluor488-labeled goat anti-mouse secondary antibody, and analyzed by flow cytometry. The mean percentage of cells positive for VEEV E2 and El (B) expression out of 10,000 events and the standard error of the means (SEM) are shown.

[30] Figs. 2A and 2B are graphs summarizing the antibody responses of vaccinated mice. Female BALB/c mice (N= 6 per group) were vaccinated three times at 3-week intervals with 25 μg of empty vector DNA plasmid or 25, 5, or 1 μg of either the VEEVW _T construct or the VEEVco plasmid delivered by i.m. EP. Serum samples obtained 3 weeks after each vaccination were assayed for total IgG anti-VEEV antibodies by ELISA and for VEEV-neutralizing antibodies by PRNT. The group mean logio ELISA (Fig. 2A) and PRNTgo (Fig. 2B) titers along with the standard error of the mean (SEM) are shown. *p < 0.05, ***p < 0.005, and *****p < 0.0001 for comparison between titers for VEEVco and VEEVW _T - [31] Figs. 3 A and 3B are graphs summarizing the antibody responses of vaccinated mice. Female BALB/c mice (N= 6 per group) were vaccinated twice at a 3-week interval with 5 μg of the VEEVco plasmid delivered by i.m. injection with and without EP. Serum samples obtained 3 weeks after each vaccination were assayed for total IgG anti-VEEV antibodies by ELISA and for VEEV-neutralizing antibodies by PR T. The group mean logio ELISA (Fig. 3A) and PRNT ₈₀ (Fig. 3B) titers along with the SEM are shown. *p < 0.05, * *p < 0.01 , and < 0.0001 for comparison between titers with and without EP.

[32] Figs. 4A and 4B are graphs summarizing the antibody responses of vaccinated mice. Female BALB/c mice (N= 10 per group) were vaccinated twice at a 3-week interval with 5 μg of empty vector DNA, VEEVCOCAP or VEEVco plasmid delivered by i.m. EP. Positive control mice (N= 10) each received a single vaccination with 0.5 ml of the live-attenuated VEEV IND vaccine TC-83 (1 x 10 ⁴ PFU) delivered by subcutaneous injection. Serum samples obtained 3 weeks after each vaccination were assayed for total IgG anti-VEEV antibodies by ELISA and for VEEV-neutralizing antibodies by PRNT. The group mean logio ELISA (Fig. 4A) and PRNT ₈₀ (Fig. 4B) titers along with the SEM are shown. **p < 0.01 , < 0.001 , and <

0.0001 for comparison between titers for VEEVCOCAP, VEEVCO, and TC-83.

[33] Fig. 5 is a graph showing the survival of vaccinated mice. Female BALB/c mice (N = 10 per group) vaccinated twice at a 3 -week interval with 5 μg of empty vector DNA, VEEVCOCAP , or VEEVco plasmid delivered by i.m. EP and positive control mice (N= 10) receiving a single vaccination with 0.5 ml of the live-attenuated VEEV IND vaccine TC-83 (1 x 10 ⁴ PFU) delivered by subcutaneous injection were challenged 4 weeks after the final vaccination with about 10 ⁴ PFU (> 1 ,000 LD ₅₀ ) of VEEV IAB by the aerosol route. Kaplan-Meier survival analysis is shown indicating the percentage of surviving mice at each day of a 28-day post-challenge observation period.

[34] Figs. 6A and 6B are graphs showing the cellular immune responses of

vaccinated mice. Pooled sera obtained on day 42 from mice vaccinated with empty vector DNA, VEEVco plasmid, and TC-83 from the challenge study were assayed for total IgG, IgGl , and IgG2 anti-VEEV antibodies by ELISA and the titers are shown (Fig. 6A). Female BALB/c mice (N= 6 per group) were vaccinated twice at a 3-week interval with 5 μg of empty vector or VEEVco plasmid delivered by i.m. EP. Two weeks after the second vaccination, splenocytes were isolated and restimulated with no peptide, Concanavalin A, pools of overlapping peptides representing the unrelated β-Galactosidase protein, or pools of overlapping peptides representing the VEEV IAB E2 or El envelope glycoproteins and analyzed by IFN-γ ELISpot. The group mean spot forming units (SFU) per 10 ⁶ cells along with the SEM are shown (Fig. 6B). *p < 0.05 for comparison between IFN-γ responses for VEEV E2 and El peptide pools.

[35] Fig. 7 shows the antibody responses of vaccinated rabbits. Female New

Zealand White rabbits (N= 5) were vaccinated with 500 μg of the VEEVco plasmid delivered by i.m. EP on days 0, 28, and 230 (indicated by arrowheads). Serum samples obtained from the rabbits on days 0, 28, 42, 230, 266, and 349 were analyzed for VEEV-neutralizing antibodies by PRNT. The group mean logio PRNTgo titers along with the SEM are shown. *p < 0.05 and < 0.005 for comparison between titers after boosting DNA vaccinations.

[36] Fig. 8 is a graph showing the antibody responses of vaccinated nonhuman primates. Adult male cynomolgus macaques (N = 4 per group) were vaccinated with 500 μg of the empty vector DNA plasmid or 500 μg or 50 μg of the VEEVco plasmid delivered by i.m. EP at days 0 and 56 (indicated by arrowheads). Serum samples obtained from the macaques on days 0, 28, 56, 84, and 1 12 were analyzed for VEEV- neutralizing antibodies by PRNT. The group mean logio PRNTgo titers along with the SEM are shown. < 0.0001 for comparison between titers after boosting DNA vaccinations.

[37] Figs. 9A, 9B and 9C are graphs showing the data for the aerosol challenge of vaccinated macaques. Adult male cynomolgus macaques (N = 4 per group) vaccinated with 500 μg of the empty vector DNA plasmid or 500 μg or 50 μg of the VEEVco plasmid delivered by i.m. EP at days 0 and 56 were challenged with a calculated dose of 3 x 10 ⁸ PFU (-300 ED ₅₀ ) of VEEV IAB by the aerosol route. After challenge, the macaques were monitored for serum viremia by plaque assay (Fig. 9A), for fever responses by telemetry (Fig. 9B), and for lymphopenia by determining PBL counts (Fig. 9C). The group mean viremias, temperature elevations, and percent changes in peripheral blood lymphocyte (PBL) counts along with the SEM are shown. *p < 0.05 and ****/? < 0.001 for comparison between the mean temperature elevations of macaques vaccinated with empty vector and VEEVco plasmid.

[38] Fig. 10A is a graph summarizing the antibody responses of vaccinated mice.

Female BALB/c mice (N = 10 per group) were vaccinated three times at a 3 -week interval with 4 μg of empty vector DNA or VEEV T plasmid or with 4 μg each of the VEEVWT _? EEEVWT, and WEEVWT plasmids delivered by PMED. Positive control mice (N = 10) each received a single vaccination with 0.5 ml of the live-attenuated VEEV IND vaccine TC-83 (1 x 10 ⁴ PFU) delivered by subcutaneous injection.

Serum samples obtained 3 weeks after each vaccination were assayed for total IgG anti-VEEV antibodies by ELISA, and the serum samples obtained 3 weeks after the last vaccination were assayed for VEEV-neutralizing antibody responses by PR T. The group mean logio ELISA and PRNTgo titers along with the SEM are shown.

[39] Fig. 1 OB is a graph showing the survival of vaccinated mice. Female BALB/c mice (N = 10 per group) vaccinated three times at a 3-week interval with 4 μg of empty vector DNA or VEEVWT plasmid or with 4 μg each of the VEEVWT _? EEEVWT, and WEEVWT plasmids delivered by PMED and positive control mice (N = 10) receiving a single vaccination with 0.5 ml of the live-attenuated VEEV IND vaccine TC-83 (1 x 10 ⁴ PFU) delivered by subcutaneous injection were challenged 4 weeks after the final vaccination with about 10 ⁴ PFU (> 1,000 LD ₅₀ ) of VEEV IAB by the aerosol route. Kaplan-Meier survival analysis is shown indicating the percentage of surviving mice at each day of a 28-day post-challenge observation period.

[40] Figs. 1 1 A and 1 IB are graphs summarizing the antibody responses of

vaccinated mice. Female BALB/c mice (N = 10 per group) were vaccinated twice at a 3 -week interval with 5 μg of empty vector DNA or VEEVco or 5 μg each of the VEEVco, EEEVco, and WEEVco plasmids delivered by i.m. EP. Positive control mice (N = 10) each received a single vaccination with 0.5 ml of the live-attenuated VEEV IND vaccine TC-83 (1 x 10 ⁴ PFU) delivered by subcutaneous injection.

Serum samples obtained 3 weeks after each vaccination were assayed for total IgG anti-VEEV antibodies by ELISA and for VEEV-neutralizing antibodies by PRNT. The group mean log ₁₀ ELISA (Fig. 1 1 A) and PRNTgo (Fig. 1 IB) titers along with the SEM are shown.

[41] Fig. 12 is a graph showing the survival of vaccinated mice. Female BALB/c mice (N = 10 per group) vaccinated twice at a 3 -week interval with 5 μg of empty vector DNA or VEEVco or 5 μg each of the VEEVco, EEEVco, and WEEVco plasmids delivered by i.m. EP and positive control mice (N = 10) receiving a single vaccination with 0.5 ml of the live-attenuated VEEV IND vaccine TC-83 (1 x 10 ⁴ PFU) delivered by subcutaneous injection were challenged 4 weeks after the final vaccination with about 10 ⁴ PFU (> 1 ,000 LD ₅₀ ) of VEEV IAB by the aerosol route. Kaplan-Meier survival analysis is shown indicating the percentage of surviving mice at each day of a 28-day post-challenge observation period.

[42] Fig. 13 is a graph showing the cellular immune responses of vaccinated mice.

Female BALB/c mice (N = 6 per group) were vaccinated twice at a 3 -week interval with 5 μg of empty vector or VEEVco plasmid or 5 μg each of the VEEVco _?

EEEVco, and WEEVco plasmids delivered by i.m. EP. Two weeks after the second vaccination, splenocytes were isolated and restimulated with no peptide,

Concanavalin A, pools of overlapping peptides representing the unrelated β- Galactosidase protein, or pools of overlapping peptides representing the VEEV IAB E2 or El envelope glycoproteins and analyzed by IFN-γ ELISpot. The mean spot forming units (SFU) per 10 ⁶ cells along with the SEM for each group are shown (Fig. 12).

[43] Fig. 14 shows the antibody responses of vaccinated rabbits. Female New

Zealand White rabbits (N= 5) were vaccinated with 500 μg of the VEEVco plasmid or 500 μg each of the VEEVco _? EEEVco, and WEEVco plasmids delivered by i.m. EP on days 0, 28, and 230 (indicated by arrowheads). Serum samples obtained from the rabbits on days 0, 28, 42, 230, 266, and 349 were analyzed for VEEV-neutralizing antibodies by PRNT. The mean logio PRNTgo titers along with the SEM for each group are shown.

[44] Fig. 15A is a graph summarizing the antibody responses of vaccinated mice.

Female BALB/c mice (N = 10 per group) were vaccinated three times at a 3 -week interval with 4 μg of empty vector DNA or EEEVWT plasmid or with 4 μg each of the EEEVWT _? VEEVWT, and WEEVWT plasmids delivered by PMED. Positive control mice (N = 10) each received a single vaccination with 0.5 ml of the formalin- inactivated EEEV IND vaccine delivered by subcutaneous injection. Serum samples obtained 3 weeks after each vaccination were assayed for total IgG anti-VEEV antibodies by ELISA, and the serum samples obtained 3 weeks after the last vaccination were also assayed for VEEV-neutralizing antibody responses by PRNT. The group mean logio ELISA and PRNTgo titers along with the SEM are shown.

[45] Fig. 15B is a graph showing the survival of vaccinated mice. Female BALB/c mice (N = 10 per group) vaccinated three times at a 3-week interval with 4 μg of empty vector DNA or EEEVWT plasmid or with 4 μg each of the EEEVWT _? VEEVWT, and WEEVWT plasmids delivered by PMED and positive control mice (N = 10) receiving a single vaccination with 0.5 ml of the formalin-inactivated EEEV IND vaccine delivered by subcutaneous injection were challenged 4 weeks after the final vaccination with about 10 ⁴ PFU (-300 LD ₅₀ ) of EEEV PE6 by the aerosol route. Kaplan-Meier survival analysis is shown indicating the percentage of surviving mice at each day of a 28-day post-challenge observation period.

[46] Figs. 16A and 16B are graphs summarizing the in vitro expression analysis in transfected cells. Radiolabeled lysates of COS-7 cells transiently trans fected with 5 μg of empty vector, EEEV _WT , or EEEVco DNA were immune precipitated with EEEV hyperimmune mouse ascitic fluids (HMAF) and analyzed by SDS-PAGE and phosphor imaging (Fig. 16A). COS-7 cells transiently transfected with 5-500 ng of empty vector, EEEVW _T , or EEEVco DNA were permeabilized, stained with an EEEV E2- or El -specific mouse mAb and a AlexaFluor488-labeled goat anti-mouse secondary antibody, and analyzed by flow cytometry. The mean percentage of cells positive for EEEV E2 and El (Fig. 16B) expression out of 10,000 events and the standard error of the means (SEM) are shown.

[47] Figs. 17A and 17B are graphs summarizing the antibody responses of

vaccinated mice. Female BALB/c mice (N= 6 per group) were vaccinated three times at 3 -week intervals with 25 μg of empty vector DNA plasmid or 25, 5, or 1 μg of either the EEEVW _T construct or the EEEVco plasmid delivered by i.m. EP. Serum samples obtained 3 weeks after each vaccination were assayed for total IgG anti- VEEV antibodies by ELISA and for VEEV-neutralizing antibodies by PRNT. The mean logio ELISA (Fig. 17A) and PRNT go (Fig. 17B) titers along with the standard error of the mean (SEM) for each group are shown.

[48] Figs. 18A and 18B are graphs summarizing the antibody responses of

vaccinated mice. Female BALB/c mice (N= 10 per group) were vaccinated twice at a 3 -week interval with 5 μg of empty vector DNA or EEEVco or 5 μg each of the EEEVco, VEEVco, and WEEVco plasmids delivered by i.m. EP. Positive control mice (N= 10) each received a single vaccination with 0.5 ml of the formalin- inactivated EEEV IND vaccine delivered by subcutaneous injection. Serum samples obtained 3 weeks after each vaccination were assayed for total IgG anti-EEEV antibodies by ELISA and for EEEV-neutralizing antibodies by PRNT. The mean logio ELISA (Fig. 18A) and PRNT ₈₀ (Fig. 18B) titers along with the SEM for each group are shown.

[49] Fig. 19 is a graph showing the survival of vaccinated mice. Female BALB/c mice (N= 10 per group) vaccinated twice at a 3 -week interval with 5 μg of empty vector DNA or EEEVco or 5 μg each of the EEEVco, VEEVco, and WEEVco plasmids delivered by i.m. EP and positive control mice (N = 10) receiving a single vaccination with 0.5 ml of the formalin-inactivated EEEV IND vaccine delivered by subcutaneous injection were challenged 4 weeks after the final vaccination with about 10 ⁴ PFU (-300 LD ₅₀ ) of EEEV FL91 -4679 by the aerosol route. Kaplan-Meier survival analysis is shown indicating the percentage of surviving mice at each day of a 28-day post-challenge observation period.

[50] Fig. 20 shows the antibody responses of vaccinated rabbits. Female New

Zealand White rabbits (N= 5) were vaccinated with 500 μg of the EEEVco plasmid or 500 μg each of the EEEVco _? VEEVco, and WEEVco plasmids delivered by i.m. EP on days 0, 28, and 230 (indicated by arrowheads). Serum samples obtained from the rabbits on days 0, 28, 42, 230, 266, and 349 were analyzed for EEEV -neutralizing antibodies by PRNT. The mean logio PRNTgo titers along with the SEM for each group are shown.

[51] Fig. 21 is a graph showing the antibody responses of vaccinated nonhuman primates. Adult cynomolgus macaques of both sexes (N= 4 per group) were vaccinated with 500 μg of the empty vector DNA plasmid, 500 μg of the EEEVco plasmid, or 500 μg each of the EEEVco _? VEEVco, and WEEVco plasmids delivered by i.m. EP at days 0, 28, and 56. Serum samples obtained from the macaques on days 0, 28, 56, and 84 were analyzed for EEEV-neutralizing antibodies by PRNT. The logio PRNTgo titers are shown along with a bar representing the mean titer for each group.

[52] Figs. 22A and 22B are graphs showing the data for the aerosol challenge of vaccinated macaques. Adult cynomolgus macaques of both sexes (N = 4 per group) vaccinated with 500 μg of the empty vector DNA plasmid, 500 μg of the EEEVco plasmid, or 500 μg each of the EEEVco _? VEEVco, and WEEVco plasmids delivered by i.m. EP on days 0, 28, and 56 were challenged with a calculated dose of 1.6 x 10 ⁸ PFU of EEEV FL91-4679 by the aerosol route. After challenge, the macaques were monitored for serum viremia by plaque assay (Fig. 22A) and for clinical signs of disease by blinded observers using established criteria (Fig. 22B). The mean viremias and clinical scores along with the SEM are shown for each group.

[53] Fig. 23A is a graph summarizing the antibody responses of vaccinated mice.

Female BALB/c mice (N = 10 per group) were vaccinated three times at a 3 -week interval with 4 μg of empty vector DNA or WEEVWT plasmid or with 4 μg each of the WEEVW _T? VEEVW _T , and EEEVW _T plasmids delivered by PMED. Positive control mice (N= 10) each received a single vaccination with 0.5 ml of the formalin- inactivated WEEV IND vaccine delivered by subcutaneous injection. Serum samples obtained 3 weeks after each vaccination were assayed for total IgG anti-WEEV antibodies by ELISA, and the serum samples obtained 3 weeks after the last vaccination were also assayed for WEEV-neutralizing antibody responses by PRNT. The group mean logio ELISA and PRNTgo titers along with the SEM are shown.

[54] Fig. 23B is a graph showing the survival of vaccinated mice. Female BALB/c mice (N= 10 per group) vaccinated three times at a 3-week interval with 4 μg of empty vector DNA or WEEVW _T plasmid or with 4 μg each of the WEEVW _T?

VEEVW _T , and EEEV _WT plasmids delivered by PMED and positive control mice (N = 10) receiving a single vaccination with 0.5 ml of the formalin-inactivated WEEV IND vaccine delivered by subcutaneous injection were challenged 4 weeks after the final vaccination with about 10 ⁴ PFU of WEEV CBA87 by the aerosol route. Kaplan- Meier survival analysis is shown indicating the percentage of surviving mice at each day of a 28-day post-challenge observation period.

[55] Fig. 24 is a graph summarizing the in vitro expression analysis in transfected cells. Radiolabeled lysates of COS-7 cells transiently transfected with 5 μg of empty vector, WEEVW _T , or WEEVco DNA were immune precipitated with WEEV hyperimmune mouse ascitic fluids (HMAF) and analyzed by SDS-PAGE and phosphor imaging.

[56] Figs. 25A and 25B are graphs summarizing the antibody responses of

vaccinated mice. Female BALB/c mice (N= 6 per group) were vaccinated three times at 3 -week intervals with 25 μg of empty vector DNA plasmid or 25, 5, or 1 μg of either the WEEVW _T construct or the WEEVco plasmid delivered by i.m. EP.

Serum samples obtained 3 weeks after each vaccination were assayed for total IgG anti-WEEV antibodies by ELISA and for WEEV-neutralizing antibodies by PRNT. The mean logio ELISA (Fig. 25 A) and PRNTgo (Fig. 25B) titers along with the standard error of the mean (SEM) for each group are shown.

[57] Figs. 26A and 26B are graphs summarizing the antibody responses of

vaccinated mice. Female BALB/c mice (N= 10 per group) were vaccinated twice at a 3 -week interval with 5 μg of empty vector DNA or WEEVco or 5 μg each of the WEEVco, VEEVco, and EEEVco plasmids delivered by i.m. EP. Positive control mice (N= 10) each received a single vaccination with 0.5 ml of the formalin- inactivated WEEV IND vaccine delivered by subcutaneous injection. Serum samples obtained 3 weeks after each vaccination were assayed for total IgG anti-WEEV antibodies by ELISA and for WEEV -neutralizing antibodies by PRNT. The mean logio ELISA (Fig. 26A) and PRNT ₈₀ (Fig. 26B) titers along with the SEM for each group are shown.

[58] Fig. 27 is a graph showing the survival of vaccinated mice. Female BALB/c mice (N = 10 per group) vaccinated twice at a 3 -week interval with 5 μg of empty vector DNA or WEEV _C0 or 5 μg each of the WEEV _C0 , VEEV _C0 , and EEEV _C0 plasmids delivered by i.m. EP and positive control mice (N = 10) receiving a single vaccination with 0.5 ml of the formalin-inactivated WEEV IND vaccine delivered by subcutaneous injection were challenged 4 weeks after the final vaccination with about 10 ⁴ PFU of WEEV CBA87 by the aerosol route. Kaplan-Meier survival analysis is shown indicating the percentage of surviving mice at each day of a 28-day post- challenge observation period.

[59] Fig. 28 shows the antibody responses of vaccinated rabbits. Female New

Zealand White rabbits (N= 5) were vaccinated with 500 μg of the WEEVco plasmid or 500 μg each of the WEEVco _? VEEVco, and EEEVco plasmids delivered by i.m. EP on days 0, 28, and 230 (indicated by arrowheads). Serum samples obtained from the rabbits on days 0, 28, 42, 230, 266, and 349 were analyzed for WEEV- neutralizing antibodies by PRNT. The mean logio PRNTgo titers along with the SEM for each group are shown.

[60] Fig. 29 is a graph showing the antibody responses of vaccinated nonhuman primates. Adult cynomolgus macaques of both sexes (N= 4 per group) were vaccinated with 500 μg of the empty vector DNA plasmid, 500 μg of the WEEVco plasmid, or 500 μg each of the WEEVco _? VEEVco, and EEEVco plasmids delivered by i.m. EP at days 0, 28, and 56. Serum samples obtained from the macaques on days 0, 28, 56, and 84 were analyzed for WEEV-neutralizing antibodies by PRNT. The logio PRNTgo titers are shown along with a bar representing the mean titer for each group.

[61] Figs. 30A, 30B and 30C schematically show the VEEVco, EEEVco, and

WEEVco plasmids and their entire sequences, respectively, as exemplified in the experiments disclosed herein.

[62] Fig. 31 shows the sequences of the plasmids exemplified in the experiments disclosed herein, where * represents the inserted EEVco sequence such that: when * is VEEVco (SEQ ID NO: l), the VEEV _C0 plasmid sequence is SEQ ID NO:7; when * is EEEVco (SEQ ID NO:3), the EEEV _C0 plasmid sequence is SEQ ID NO:8; and when * is WEEVco (SEQ ID NO:5), the WEEV _C0 plasmid sequence is SEQ ID NO:9. Although not provided in the sequence listing, it is noted that the sequences of the corresponding EEVCOCA _P plasmids merely contain the given EEVCOCA _P sequence where * is indicated.

[63] DETAILED DESCRIPTION OF THE INVENTION

[64] The present invention provides nucleic acid molecules and constructs which may be used as vaccines against VEEV, EEEV, and/or WEEV, and compositions and methods for vaccinating subjects, such as human subjects, against VEEV, EEEV, and/or WEEV.

[65] The nucleic acid molecules and constructs according to the present invention comprise codon-optimized sequences that encode structural proteins of EEVs and exclude sequences that encode the capsid proteins of EEVs.

[66] A. VEEV

[67] As exemplified herein, a wild-type VEEV DNA plasmid (VEEVW _T ) was constructed by inserting the cloned 26S structural genes (C-E3-E2-6K-E1) of VEEV subtype IAB strain Trinidad donkey (Genbank accession number L01442) behind the cytomegalovirus immediate early promoter of the eukaryotic expression vector pWRG7077, as previously described. See Riemenschneider et al. (2003) Vaccine 21 :4071-80, which is herein incorporated by reference. Codon optimized VEEV subtype IAB structural genes with the capsid gene (VEEVCOCA _P ) and lacking the capsid gene (VEEVco) were generated by subjecting the cloned wild type 26S structural genes (C-E3-E2-6K-E1) and the cloned wild type 26S structural genes lacking the capsid sequence (E3-E2-6K-E1), respectively, of VEEV subtype IAB strain Trinidad donkey (Genbank accession number L01442) to a GeneOptimizer™ bioinformatic algorithm (Geneart, Regensburg, Germany) for mammalian expression and then synthesized using methods known in the art. DNA plasmids were constructed by cloning the synthesized codon optimized genes into the Notl and Bglll restriction sites of pWRG7077 using methods known in the art.

[68] The sequences of the nucleic acid molecules are as follows:

[69] VEEVco: ATGAGCCTGGTGACCACCATGTGCCTGCTGGCCAACGTGACCT TCCCCTGCGCCCAG CCCCCCATCTGCTACGACCGGAAGCCCGCCGAGACCCTGGCCATGCTGTCCGTGAAC GTGGACAACCCCGGCTACGACGAGCTGCTGGAAGCCGCCGTGAAGTGCCCCGGCAGG AAGCGGCGGAGCACCGAGGAACTGT TCAAAGAGTACAAGCTGACCCGGCCCTACATG GCCCGGTGCATCAGATGCGCCGTGGGCAGCTGCCACAGCCCCATCGCCATCGAGGCC GTGAAGAGCGACGGCCACGACGGCTACGTGCGGCTGCAGACCAGCAGCCAGTACGGC CTGGACAGCAGCGGCAACCTGAAGGGCCGGACCATGAGATACGACATGCACGGCACC ATCAAAGAGATCCCCCTGCACCAGGTGTCCCTGCACACCAGCCGGCCCTGCCACATC GTGGACGGCCACGGCTACT T TCTGCTGGCCAGGTGCCCTGCCGGCGACAGCATCACC ATGGAAT TCAAGAAAGACAGCGTGACCCACAGCTGCAGCGTGCCCTACGAGGTGAAG T TCAACCCCGTGGGCCGGGAGCTGTACACCCACCCCCCCGAGCACGGCGTGGAGCAG GCCTGCCAGGTGTACGCCCACGACGCCCAGAACAGGGGCGCCTACGTGGAGATGCAC CTGCCCGGCAGCGAGGTGGACAGCTCCCTGGTGTCCCTGAGCGGCAGCAGCGTGACC GTGACCCCCCCTGTGGGCACCAGCGCCCTGGTGGAGTGCGAGTGCGGCGGCACCAAG ATCAGCGAGACCATCAACAAGACCAAGCAGT TCAGCCAGTGCACCAAGAAAGAGCAG TGCCGGGCCTACCGGCTGCAGAACGACAAGTGGGTGTACAACAGCGACAAGCTGCCC AAAGCCGCCGGAGCCACCCTGAAGGGCAAGCTGCACGTGCCT T T TCTGCTGGCTGAC GGCAAGTGCACCGTGCCCCTGGCCCCCGAGCCCATGATCACCT TCGGCT TCAGAAGC GTGAGCCTGAAGCTGCACCCCAAGAACCCCACCTACCTGACCACCCGGCAGCTGGCC GATGAGCCCCACTACACCCACGAGCTGATCAGCGAGCCCGCCGTGCGGAACT TCACC GTGACCGAGAAGGGCTGGGAGT TCGTGTGGGGCAACCACCCCCCCAAGAGGT TCTGG GCTCAGGAAACAGCCCCTGGCAACCCCCACGGCCTGCCTCACGAGGTGATCACCCAC TACTACCACAGATACCCCATGAGCACCATCCTGGGCCTGAGCATCTGCGCCGCCATC GCCACCGTGAGCGTGGCCGCCAGCACCTGGCTGT TCTGCCGGTCCCGGGTGGCCTGC CTGACCCCCTACAGGCTGACCCCCAACGCCCGGATCCCCT TCTGCCTGGCCGTGCTG TGCTGCGCCCGGACCGCCAGAGCCGAGACCACCTGGGAGAGCCTGGACCACCTGTGG AACAACAACCAGCAGATGT TCTGGATCCAGCTGCTGATCCCCCTGGCCGCCCTGATC GTGGTGACCCGGCTGCTGAGATGCGTGTGCTGCGTGGTGCCCT TCCTGGTGATGGCC GGGGCTGCAGGGGCCGGCGCCTATGAGCACGCCACCACCATGCCCAGCCAGGCCGGC ATCAGCTACAACACCATCGTGAACAGGGCCGGCTACGCCCCCCTGCCCATCAGCATC ACCCCTACCAAGATCAAGCTGATCCCCACCGTGAACCTGGAATACGTGACCTGCCAC TACAAGACCGGCATGGACAGCCCCGCCATCAAGTGCTGCGGCAGCCAGGAATGCACC CCCACCTACAGGCCCGACGAGCAGTGCAAGGTGT TCACCGGCGTGTACCCCT TCATG TGGGGCGGAGCCTACTGCT TCTGCGACACCGAGAACACCCAGGTGTCCAAGGCCTAC GTGATGAAGTCCGACGAT TGCCTGGCCGACCACGCCGAGGCCTACAAGGCCCACACC GCCAGCGTGCAGGCCT TCCTGAACATCACCGTGGGCGAGCACAGCATCGTGACCACC GTGTACGTGAACGGCGAGACCCCCGTGAACT TCAACGGCGTGAAGCTGACCGCCGGA CCCCTGAGCACCGCCTGGACCCCCT TCGACCGGAAGATCGTGCAGTACGCCGGCGAA ATCTACAACTACGACT TCCCCGAGTATGGCGCCGGACAGCCTGGCGCCT TCGGCGAC ATCCAGAGCCGGACCGTGAGCAGCAGCGACCTGTACGCCAACACCAACCTGGTGCTG CAGCGGCCCAAGGCCGGAGCCATCCACGTGCCCTACACCCAGGCCCCCAGCGGCT TC GAGCAGTGGAAGAAGGACAAGGCCCCCTCCCTGAAGT TCACCGCCCCCT TCGGCTGT GAAATCTACACCAACCCCATCCGGGCCGAGAACTGTGCCGTGGGCTCCATCCCTCTG GCCT TCGACATCCCCGACGCCCTGT TCACCAGAGTGTCCGAGACCCCCACCCTGTCT GCCGCCGAGTGCACCCTGAACGAGTGCGTCTACTCCTCTGACT TCGGCGGCATCGCC ACAGTGAAGTACAGCGCCAGCAAGAGCGGCAAGTGTGCCGTGCACGTGCCCAGCGGC ACAGCCACACTGAAGGAAGCCGCCGTGGAGCTGACCGAGCAGGGCAGCGCCACCATC CACT TCAGCACCGCCAACATCCACCCCGAGT TCAGGCTGCAGAT T TGCACCAGCTAC GTGACATGCAAGGGCGACTGCCACCCCCCTAAGGACCACATCGTGACCCACCCCCAG TACCACGCCCAGACCT TCACAGCCGCCGTGTCCAAGACAGCCTGGACCTGGCTGACC AGCCTGCTGGGCGGCAGCGCCGTGATCATCATCATCGGCCTGGTGCTGGCCACCATC GTGGCCATGTACGTGCTGACCAACCAGAAACACAACTGATGA (SEQ ID NO: l). As used herein, a plasmid containing SEQ ID NO: l is referred to as "VEEVco plasmid".

[70] VEEVCOCAP:

ATGTTCCCATTCCAGCCCATGTACCCCATGCAGCCCATGCCCTACCGGAACCCCTTT GCCGCCCCTCGGAGGCCCTGGTTCCCCCGGACCGACCCCTTCCTGGCCATGCAGGTG CAGGAACTGACCAGAAGCATGGCCAACCTGACCTTCAAGCAGCGGCGGGACGCCCCT CCTGAGGGCCCCTCCGCCAAGAAGCCCAAGAAAGAGGCCAGCCAGAAGCAGAAGGGC GGAGGGCAGGGCAAGAAGAAGAAAAACCAGGGGAAGAAGAAAGCCAAGACCGGCCCT CCCAACCCCAAGGCCCAGAACGGCAACAAGAAAAAGACCAACAAGAAGCCCGGCAAG CGGCAGCGGATGGTGATGAAGCTGGAAAGCGACAAGACCTTCCCCATCATGCTGGAA GGCAAGATCAACGGCTACGCCTGCGTGGTGGGCGGCAAGCTCTTTAGACCCATGCAC GTGGAGGGCAAAATTGACAACGACGTGCTGGCCGCCCTGAAAACCAAGAAGGCCAGC AAGTACGACCTGGAATACGCCGACGTGCCCCAGAACATGCGGGCCGACACCTTCAAG TACACCCACGAGAAGCCCCAGGGCTACTACAGCTGGCACCACGGAGCCGTGCAGTAC GAGAACGGCCGGTTCACCGTGCCCAAGGGCGTCGGCGCCAAGGGCGACAGCGGCAGA CCCATCCTGGACAACCAGGGCCGGGTGGTGGCCATCGTGCTGGGCGGCGTGAACGAG GGCAGCCGGACCGCCCTGAGCGTGGTGATGTGGAACGAGAAGGGCGTGACCGTGAAG TACACCCCTGAGAACTGCGAGCAGTGGAGCCTGGTGACCACCATGTGCCTGCTGGCC AACGTGACCTTCCCCTGCGCCCAGCCCCCCATCTGCTACGACCGGAAGCCCGCCGAG ACCCTGGCCATGCTGTCCGTGAACGTGGACAACCCCGGCTACGACGAGCTGCTGGAA GCCGCCGTGAAGTGCCCCGGCAGGAAGCGGCGGAGCACCGAGGAACTGTTCAAAGAG TACAAGCTGACCCGGCCCTACATGGCCCGGTGCATCAGATGCGCCGTGGGCAGCTGC CACAGCCCCATCGCCATCGAGGCCGTGAAGAGCGACGGCCACGACGGCTACGTGCGG CTGCAGACCAGCAGCCAGTACGGCCTGGACAGCAGCGGCAACCTGAAGGGCCGGACC ATGAGATACGACATGCACGGCACCATCAAAGAGATCCCCCTGCACCAGGTGTCCCTG CACACCAGCCGGCCCTGCCACATCGTGGACGGCCACGGCTACTTTCTGCTGGCCAGG TGCCCTGCCGGCGACAGCATCACCATGGAATTCAAGAAAGACAGCGTGACCCACAGC TGCAGCGTGCCCTACGAGGTGAAGTTCAACCCCGTGGGCCGGGAGCTGTACACCCAC CCCCCCGAGCACGGCGTGGAGCAGGCCTGCCAGGTGTACGCCCACGACGCCCAGAAC AGGGGCGCCTACGTGGAGATGCACCTGCCCGGCAGCGAGGTGGACAGCTCCCTGGTG TCCCTGAGCGGCAGCAGCGTGACCGTGACCCCCCCTGTGGGCACCAGCGCCCTGGTG GAGTGCGAGTGCGGCGGCACCAAGATCAGCGAGACCATCAACAAGACCAAGCAGTTC AGCCAGTGCACCAAGAAAGAGCAGTGCCGGGCCTACCGGCTGCAGAACGACAAGTGG GTGTACAACAGCGACAAGCTGCCCAAAGCCGCCGGAGCCACCCTGAAGGGCAAGCTG CACGTGCCTTTTCTGCTGGCTGACGGCAAGTGCACCGTGCCCCTGGCCCCCGAGCCC ATGATCACCTTCGGCTTCAGAAGCGTGAGCCTGAAGCTGCACCCCAAGAACCCCACC TACCTGACCACCCGGCAGCTGGCCGATGAGCCCCACTACACCCACGAGCTGATCAGC GAGCCCGCCGTGCGGAACTTCACCGTGACCGAGAAGGGCTGGGAGTTCGTGTGGGGC AACCACCCCCCCAAGAGGTTCTGGGCTCAGGAAACAGCCCCTGGCAACCCCCACGGC CTGCCTCACGAGGTGATCACCCACTACTACCACAGATACCCCATGAGCACCATCCTG GGCCTGAGCATCTGCGCCGCCATCGCCACCGTGAGCGTGGCCGCCAGCACCTGGCTG TTCTGCCGGTCCCGGGTGGCCTGCCTGACCCCCTACAGGCTGACCCCCAACGCCCGG ATCCCCTTCTGCCTGGCCGTGCTGTGCTGCGCCCGGACCGCCAGAGCCGAGACCACC TGGGAGAGCCTGGACCACCTGTGGAACAACAACCAGCAGATGTTCTGGATCCAGCTG CTGATCCCCCTGGCCGCCCTGATCGTGGTGACCCGGCTGCTGAGATGCGTGTGCTGC GTGGTGCCCTTCCTGGTGATGGCCGGGGCTGCAGGGGCCGGCGCCTATGAGCACGCC ACCACCATGCCCAGCCAGGCCGGCATCAGCTACAACACCATCGTGAACAGGGCCGGC TACGCCCCCCTGCCCATCAGCATCACCCCTACCAAGATCAAGCTGATCCCCACCGTG AACCTGGAATACGTGACCTGCCACTACAAGACCGGCATGGACAGCCCCGCCATCAAG TGCTGCGGCAGCCAGGAATGCACCCCCACCTACAGGCCCGACGAGCAGTGCAAGGTG T TCACCGGCGTGTACCCCT TCATGTGGGGCGGAGCCTACTGCT TCTGCGACACCGAG

AACACCCAGGTGTCCAAGGCCTACGTGATGAAGTCCGACGAT TGCCTGGCCGACCAC

GCCGAGGCCTACAAGGCCCACACCGCCAGCGTGCAGGCCT TCCTGAACATCACCGTG

GGCGAGCACAGCATCGTGACCACCGTGTACGTGAACGGCGAGACCCCCGTGAACT TC

AACGGCGTGAAGCTGACCGCCGGACCCCTGAGCACCGCCTGGACCCCCT TCGACCGG

AAGATCGTGCAGTACGCCGGCGAAATCTACAACTACGACT TCCCCGAGTATGGCGCC

GGACAGCCTGGCGCCT TCGGCGACATCCAGAGCCGGACCGTGAGCAGCAGCGACCTG

TACGCCAACACCAACCTGGTGCTGCAGCGGCCCAAGGCCGGAGCCATCCACGTGCCC

TACACCCAGGCCCCCAGCGGCT TCGAGCAGTGGAAGAAGGACAAGGCCCCCTCCCTG

AAGT TCACCGCCCCCT TCGGCTGTGAAATCTACACCAACCCCATCCGGGCCGAGAAC

TGTGCCGTGGGCTCCATCCCTCTGGCCT TCGACATCCCCGACGCCCTGT TCACCAGA

GTGTCCGAGACCCCCACCCTGTCTGCCGCCGAGTGCACCCTGAACGAGTGCGTCTAC

TCCTCTGACT TCGGCGGCATCGCCACAGTGAAGTACAGCGCCAGCAAGAGCGGCAAG

TGTGCCGTGCACGTGCCCAGCGGCACAGCCACACTGAAGGAAGCCGCCGTGGAGCTG

ACCGAGCAGGGCAGCGCCACCATCCACT TCAGCACCGCCAACATCCACCCCGAGT TC

AGGCTGCAGAT T TGCACCAGCTACGTGACATGCAAGGGCGACTGCCACCCCCCTAAG

GACCACATCGTGACCCACCCCCAGTACCACGCCCAGACCT TCACAGCCGCCGTGTCC

AAGACAGCCTGGACCTGGCTGACCAGCCTGCTGGGCGGCAGCGCCGTGATCATCATC

ATCGGCCTGGTGCTGGCCACCATCGTGGCCATGTACGTGCTGACCAACCAGAAACAC

AACTGATGA (SEQ ID N0:2). As used herein, a plasmid containing SEQ ID NO:2 is referred to as "VEEVCOCAP plasmid".

The live-attenuated VEEV vaccine, TC-83, was manufactured by the National Drug Company (Philadelphia, PA).

To evaluate the protective efficacy of VEEVco (SEQ ID NO: 1) as a DNA vaccine, an established and well-characterized aerosol challenge model of VEEV infection in cynomolgus macaques was used. See Pratt et al. (2003) Vaccine

21 :3854-62; Pratt et al. (1998) Vaccine 16: 1056-64; Reed et al. (2005) Vaccine 23:3139-47; and Reed et al. (2004) J Infect Dis 189: 1013-7. Previously, it was found that cynomolgus macaques vaccinated with three doses of VEEV _WT DNA delivered by PMED developed low levels of VEEV-neutralizing antibodies and were only partially protected from aerosol VEEV challenge. See Dupuy et al. (2010) Vaccine 28:7345-50. As disclosed herein, it was unexpectedly found that cynomolgus macaques receiving two EP vaccinations with a dose as low as 50 μg of VEEVco plasmid developed increased VEEV-neutralizing antibody titers as compared to macaques receiving the VEEVW _T plasmid by PMED. In addition, these macaques had no detectable serum viremia and had reduced febrile reactions, lymphopenia, and clinical signs of disease as compared to negative control macaques after aerosol VEEV challenge. The macaques vaccinated with the VEEVco plasmid displayed no neurological signs of disease after challenge. Thus, the present invention provides a nucleic acid vaccine that, at relatively low doses, provides protection against viremia and manifestation of disease symptoms that is at least similar to that provided by TC- 83 and C-84.

[73] METHODS

[74] IN VITRO EXPRESSION ANALYSIS. Transient transfection, radiolabeling, and immune precipitation were performed as described previously. See Schmaljohn et al. (1997) J Virol 71 :9563-9. Briefly, COS-7 cells grown in complete DMEM

(Invitrogen, Carlsbad, CA) in 25-cm ² culture flasks at 37°C with 5% C0 ₂ were transfected with 5 μg of the appropriate DNA plasmid by using FuGene 6 reagent (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's

recommendations. After 24 h, the cells were starved for 30 min in methionine- and cysteine-free medium and then radiolabeled for 4 hours in medium containing 200 μθ of Promix ([ ³⁵ S]methionine and [ ³⁵ S]cysteine; Amersham Biosciences,

Piscataway, NJ) per 25-cm ² flask. Cell ly sates were made using 4% Zwittergent (Calbiochem, San Diego, CA) buffer containing Complete Protease Inhibitor Cocktail (Roche), and the lysates were normalized for protein concentration by BCA protein assay (Pierce Biotechnology, Rockford, IL). Immune precipitation of 200 μΐ of labeled lysate was performed with 10 μΐ of VEEV IAB hyperimmune mouse ascitic fluids and 150 μΐ of Protein G-Sepharose (Sigma- Aldrich, St. Louis, MO). Reduced samples (25 μΐ each) were analyzed by SDS-PAGE on a 4-12% Bis-Tris gradient gel (Invitrogen) by using MOPS running buffer (Invitrogen) at 200 V. The gel was fixed, dried, and analyzed using a Cyclone phosphorimager and OptiQuant software (PerkinElmer, Waltham, MA).

[75] Flow cytometry of transiently transfected cells was performed as described previously. See Badger et al. (201 1) Vaccine 29:6728-35. Briefly, COS-7 cells were transfected with 5-250 ng of the appropriate DNA plasmid as described above. After 40 h, the cells were washed with PBS (Invitrogen) and detached by using trypsin- EDTA (Sigma- Aldrich). The detached cells were washed three times in 4°C FACS buffer (2% FBS and 0.1% Sodium Azide in lx PBS) and fixed by adding BD Cytofix buffer (Becton-Dickinson, Franklin Lakes, NJ) and incubating at 4°C for 15 min. The fixed cells were washed as described above and permeabilized by adding BD

Perm/Wash buffer (Becton-Dickinson) and incubating at 4°C for 15 min. The permeabilized cells were washed as described above and a 1 : 100 dilution of the 1A4A-1 VEEV E2-specific or the 3B2A9 El-specific mouse monoclonal antibody (mAb) (kind gifts of Dr. John T. Roehrig, Centers for Disease Control, Fort Collins, CO) was added to the cells. After incubation at 4°C for 30 min, the cells were washed as described above and a 1 :400 dilution of AlexaFluor488-labeled goat anti-mouse secondary antibody (Invitrogen) was added to the cells. After incubation at 4°C for 20 min, the stained cells were washed as described above and analyzed on a

FACSCalibur flow cytometer (Becton-Dickinson) to determine the percentage of cells positive for VEEV E2 or El envelope glycoprotein expression per 10,000 events.

[76] ANIMALS AND VACCINATIONS. Female BALB/c mice (6-8 weeks old) were vaccinated with plasmid solutions, i.e. VEEVco plasmid, VEEVCOCA _P plasmid, and VEEVW _T plasmid, diluted to the appropriate concentration in calcium- and magnesium-free PBS (Invitrogen) by i.m. EP using the TriGrid™ Delivery System (TDS) (Ichor Medical Systems, San Diego, CA) as described previously. See Luxembourg et al. (2008) Vaccine 26:4025-33. Briefly, mice anesthetized with i.m. injection of a diluted acepromazine/ketamine/xylazine mixture or with isofluorane gas were injected into one tibialis anterior muscle with 20 μΐ of a plasmid solution using a 3/10 cm ³ U-100 insulin syringe (Becton-Dickinson) inserted into the center of a TriGrid™ electrode array with 2.5 mm electrode spacing. Injection of the plasmid solution was followed immediately by electrical stimulation at amplitude of 250 V/cm, and the total duration was 40 ms over a 400 ms interval. Some control vaccinations consisted of i.m. injection of the plasmid solutions as described above without the application of electrical stimulation. The live-attenuated VEEV IND vaccine TC-83 was delivered by subcutaneous injection.

[77] Female New Zealand White rabbits (3-3.5 kg) were anesthetized with

isofluorane gas and vaccinated with the plasmid solutions diluted to the appropriate concentration by i.m. EP with the Ichor TDS essentially as described above for mice. However, rabbits were injected with 0.5 ml of plasmid solution into one quadriceps muscle with a 1-cm ³ syringe (Becton-Dickinson) with a 23 -gauge needle, and a TriGrid electrode array with 6.0-mm electrode spacing was used.

[78] Healthy adult male cynomolgus macaques (> 5 kg) obtained from the

nonhuman primate colony at USAMRIID lacking serum neutralizing antibodies against VEEV subtype IAB, VEEV subtype IE, VEEV subtype IIIA, and EEEV and WEEV as determined by plaque reduction neutralization tests (PRNT) (as described herein) were anesthetized by i.m. injection of Telazol and vaccinated with plasmid solution diluted to the appropriate concentration by i.m. EP with the Ichor TDS as described above for rabbits.

[79] All animal research was conducted in compliance with the Animal Welfare

Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the "Guide for the Care and Use of Laboratory Animals," Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, National Academy Press, Washington, DC, 1996. The USAMRIID facility where this animal research was conducted is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International.

[80] IMMUNOLOGICAL ASSAYS. At various times after vaccination, blood samples were collected from anesthetized mice by retro-orbital bleed, from anesthetized rabbits by central auricular artery bleed, and from anesthetized macaques by femoral vein bleed, and serum was recovered by centrifugation. Total IgG anti-VEEV endpoint antibody titers were determined for serum samples by standard ELISA using sucrose-purified, irradiated whole VEEV IAB antigen as described previously. See Hodgson et al. (1999) Vaccine 17: 1151-60. Briefly, two-fold serial dilutions of sera starting at 1 : 100 were incubated with 250 ng per well of antigen in 96-well plates. Horseradish Peroxidase (HRP)-conjugated anti-mouse IgG or anti-rabbit IgG secondary antibodies (Sigma- Aldrich) and ABTS peroxidase substrate (KPL,

Gaithersburg, MD) were used for detection. For antibody isotyping ELISA, HRP- conjugated anti-mouse IgGl and anti-mouse IgG2a secondary antibodies (Bethyl Laboratories, Montgomery, TX) were used. The optical density at 405 nm was determined using a SpectraMax M2e microplate reader (Molecular Devices,

Sunnyvale, CA) and the endpoint titers were calculated using Softmax Pro v5

(Molecular Devices).

[81] The VEEV IAB-neutralizing antibody titers were determined for serum

samples by PRNT as described previously. See Hodgson (1999). Briefly, two-fold serial dilutions of sera were mixed with equal volumes of medium containing 200 PFU of virus and incubated for 24 hours at 4°C. The virus/antibody mixtures were then used to infect confluent monolayers of Vero cells contained in six-well plates for 1 hour at 37°C after which an overlay consisting of 0.6% agar in complete EBME (Invitrogen) without phenol red was added. The plates were stained 24 hours later by the addition of an overlay containing 5% neutral red and 0.6% agar in complete EBME without phenol red, and the plaques were counted 24 hours after staining. The antibody titers required for 80% reduction in the number of plaques (PRNT ₈₀ ) were then calculated for each serum sample.

[82] Anti-VEEV cellular immune responses were analyzed by IFN-γ ELISPOT assay using standard methods as described previously. See Taguchi et al. (1990) J Immunol Methods 128:65-73. Briefly, splenocytes isolated from individual spleens obtained from vaccinated mice using BD Falcon 100 μΜ nylon cell strainers (Becton- Dickinson) were resuspended in complete RPMI 1640 medium (Mediatech,

Manassas, VA). The resuspended splenocytes from each spleen were then added at a concentration of 2 x 10 ⁵ cells per well to triplicate wells of MultiScreennTs IP 0.45 μιη PVDF filter 96-well plates (Millipore, Billerica, MA) previously coated with mouse IFN-γ ELISPOT capture antibody (Becton-Dickinson). The splenocytes were then cultured with no peptide, 10 μg/ml of Concanavalin A (Sigma-Aldrich), 20 μg/ml of β-galactosidase peptide TPHPARIGL (New England Peptide, Gardner, MA), or 10 μg/ml of pooled 15-mer peptides with an 11 -base overlap spanning the VEEV IAB E2 or El envelope glycoprotein (Pepscan, Lelystad, The Netherlands) for 24 hours at 37°C with 5% C0 ₂ . Secreted IFN-γ was detected by aspirating the cell suspension and successively incubating the plate for 2 hour at room temperature with mouse IFN-γ ELISPOT detection antibody (Becton-Dickinson), for 1 hour at room temperature with streptavidin-HRP (Becton-Dickinson), and for 20 minutes at room temperature with AEC substrate (Becton-Dickinson). The substrate reaction was then stopped by washing the plates with deionized H ₂ 0, the plates were dried for 2 hours at room temperature, and the spots were enumerated.

[83] AEROSOL CHALLENGE OF MICE. Mice were placed into a Class III biological safety cabinet located inside a biosafety level-3 containment suite and exposed in a whole-body aerosol chamber to a VEEV aerosol created by a Collison nebulizer for 10 minutes as previously described. See Hart et al. (1997) Vaccine 15:363-9. VEEV subtype IAB (strain Trinidad donkey) was diluted to an appropriate starting concentration in Hank's balanced salt solution (HBSS) containing 1% fetal bovine serum for use in aerosol generation. Samples collected from the all-glass impinger (AGI) attached to the aerosol chamber were analyzed by plaque assay on Vera cells using standard methods as previously described to determine the inhaled dose of VEEV. See Pratt et al. (1998) Vaccine 16: 1056-64. The mice were monitored twice daily for clinical signs of illness and death for 28 days post-challenge and any moribund animals were euthanized. After the post-challenge observation period was completed, Kaplan-Meier survival analysis of the protection data was performed.

[84] AEROSOL CHALLENGE OF MACAQUES. Before aerosol exposure, macaques were anesthetized by i.m. injection of 6 mg/kg of Telazol and a whole-body plethysmograph was taken for 3 minutes to determine the respiratory capacity of the animal. The macaques were then inserted into a Class III biological safety cabinet located inside a biosafety level-3 containment suite and exposed in a head-only aerosol chamber to a VEEV aerosol created by a Collison nebulizer for 10 minutes as previously described. See Pratt (1998) and Reed et al. (2004) J Infect Dis 189: 1013- 7. VEEV IAB (strain Trinidad donkey) was diluted to an appropriate starting concentration in HBSS containing 1% fetal bovine serum for use in aerosol generation. Samples collected from the all-glass impinger (AGI) attached to the aerosol chamber were analyzed by plaque assay on Vero cells using standard methods as described above to determine the inhaled dose of VEEV. The macaques were monitored at least twice daily for clinical signs of illness for 28 days post-challenge. The macaques were observed for neurological signs of infection, changes in activity and behavior, and response to stimuli using predetermined criteria. The observers were blinded and were not aware of which macaques belonged to which groups.

[85] On days -3 to -1 relative to aerosol exposure and for 7 days post-challenge, the macaques were anesthetized with 3 mg/kg of Telazol and blood samples were collected to assess viremia and lymphopenia. Serum viremia was measured by plaque assay as described above. Complete blood counts were determined with a Coulter T- series instrument (Beckman Coulter, Brea, CA) and a manual differential count.

[86] TA10TA-D70 radiotelemetry implants (Data Sciences International, St. Paul,

MN) were surgically implanted subcutaneously on the dorsal surface and macaques were allowed > 30 days to recover from surgery and to acclimate before VEEV exposure. Body temperatures were recorded every 15 minutes by using the

DataQuest A.R.T. 2.1 System (Data Sciences International). Temperature monitoring was initiated 14 days before VEEV exposure to develop baseline temperature data to fit an autoregressive integrated moving average (ARIMA) model, as previously described. See Pratt (1998) and Reed (2004). Forecasted values for the post-challenge time periods were based on the training model extrapolated forward in time. Residual temperature changes after exposure were determined by subtracting the predicted temperature from the actual temperature recorded for each point. [87] STATISTICAL ANALYSIS. Logio transformations were applied to whole-virus

ELISA titers and PRNTgo titers. Mixed model analysis of variance (ANOVA) with Tukey's post hoc tests was used to compare titers between groups at each time point. Kaplan Meier survival analysis was performed for the mouse challenge study data, with log rank tests used for comparison of survival curves. The effects of whole-virus ELISA titers and PR T ₈₀ titers on the probability of survival were assessed using a backwards-selection logistic regression model. Analyses were conducted using SAS v9.2 (SAS Institute, Cary, NC).

[88] RESULTS

[89] IN VITRO EXPRESSION ANALYSIS. TO determine if the VEEV _C o vaccine

construct showed improved expression of the envelope glycoproteins as compared to the previous VEEVW _T construct, the proteins expressed in transiently transfected mammalian cells were detected by immune precipitation assay. The expression of the E2 and El envelope glycoproteins from the VEEVco DNA was increased

approximately fivefold relative to that of the VEEVW _T construct in this assay (Fig. 1 A). To further quantify the relative in vitro expression levels of these constructs, the percent of cells positive for E2 and El expression after transient trans fection of 5-250 ng of each DNA vaccine plasmid was determined by flow cytometry. In this assay, the potential impact of codon optimization of the capsid protein was also examined by including the VEEVCOCA _P plasmid. A significant increase in the mean percent of cells positive for E2 and El expression was observed for the VEEVco DNA plasmid at all concentrations except for the 5-ng sample for both E2 and El and for the 25 -ng sample for El (p < 0.05) (Fig. IB). It was unexpectedly found that the percentage of cells positive for E2 and El expression were lower for VEEVCOCA _P as compared to VEEVW _T for the 50-250 ng samples.

[90] IMMUNOGENICITY IN MICE. To determine whether i.m. EP delivery is an

effective mode of administration for the VEEVco plasmid, groups of six female BALB/c mice were vaccinated by i.m. EP three times at 3-week intervals with 25 μg, 5 μg, or 1 μg of the VEEVco plasmid, or for comparison, with the vaccine used in our earlier studies, which expresses the capsid gene as well as the envelope protein genes of wild-type VEEV (VEEVW _T ). A negative control group received the plasmid vector with no insert. [91] Serum samples obtained 3 weeks after each vaccination were assayed for total anti-VEEV IgG antibodies by ELISA and for VEEV-neutralizing antibodies by PR T. The mean ELISA titers of mice vaccinated with the VEEVco plasmid were significantly higher than those of mice vaccinated with the VEEVW _T construct for the 5 μg dose groups after a single vaccination and for the 1 μg dose groups after each of the three vaccinations (Fig. 2A). After two or three vaccinations with the 5 μg dose and after each of the three vaccinations with the 25 μg dose, the mice receiving these two plasmids displayed similar mean ELISA titers (p > 0.05). Within each group, there was no significant increase in the mean titers observed after two or three vaccinations.

[92] The mean PRNTgo titer elicited by mice vaccinated with the VEEVco plasmid was significantly higher than that observed for mice vaccinated with the VEEVW _T construct for all dose groups at all days (p < 0.01-0.0001) (Fig. 2B). Interestingly, the mean PRNTgo titer of mice vaccinated with 1 μg of the VEEVco plasmid was higher than that of mice receiving 25 μg of the VEEVW _T construct after each of the three vaccinations. As observed for the total IgG antibody titers, there was only a small difference in the level of antibodies measured by PRNT after two or three

vaccinations.

[93] To directly assess the effect of EP, a separate experiment was conducted in which groups of six female BALB/c mice were vaccinated twice at a 3 -week interval with 5 μg of the VEEVco plasmid delivered by i.m. injection with and without EP. After the second vaccination, both the mean ELISA (Fig. 3A) and PRNT (Fig. 3B) antibody titers were significantly higher in mice receiving the vaccine with EP as compared to those receiving it by injection only.

[94] VEEVco vs. VEEVCOCA _P : AS a first step toward assessing the protective efficacy of the VEEVco plasmid delivered by i.m. EP, a challenge study was performed in mice. In this study, the potential contribution of the capsid protein was also examined by including a codon optimized construct expressing all of the structural proteins of VEEV including capsid (VEEVCOCA _P plasmid). Groups of 10 female BALB/c mice were vaccinated twice at a 3 -week interval with 5 μg of these codon optimized plasmid or with 5 μg of empty vector by i.m. EP. A positive control group was also included in this experiment in which the mice were given a single subcutaneous injection of the live-attenuated VEEV IND vaccine, TC-83 (1 x 10 ⁴ PFU). [95] There was no statistical difference in the mean ELISA titers observed for serum samples collected 3 weeks after one or two vaccinations with the VEEVco plasmid or VEEVCOCA _P plasmid; however, for both of these groups, the second vaccination significantly boosted the antibody titer as compared to that measured after one vaccination (Fig. 4A). Comparing the overall antibody responses elicited by the VEEVco plasmid and the VEEVCOCA _P plasmid to that resulting from vaccination TC- 83 revealed that the VEEVco plasmid and TC-83 elicited similar titers after a single vaccination, whereas two vaccinations with the VEEVCOCA _P plasmid were required to match that obtained with TC-83. While the mean neutralizing antibody titers of mice receiving two vaccinations with the VEEVco plasmid were similar to those of mice vaccinated with TC-83, those of mice receiving two vaccinations with the

VEEVCOCA _P plasmid remained significantly lower than those vaccinated with TC-83 (Fig. 4B).

[96] To assess vaccine efficacy, the mice from all groups were challenged four weeks after the final vaccination with about 10 ⁴ PFU (> 1,000 LD ₅₀ ) of VEEV IAB strain Trinidad donkey administered by the aerosol route. Control mice, which were vaccinated with the empty vector, all displayed signs of illness after challenge including ruffled fur, loss of appetite, inactivity, and hunched backs, and all died or were euthanized due to morbidity (Fig. 5). Consistent with results from previous aerosol challenge studies (Dupuy et al. (2009) Vaccine 27:4152-60; and

Riemenschneider et al. (2003) Vaccine 21 :4071-80), 90% of the mice receiving TC- 83 survived the challenge, and the single mouse that did not survive the challenge did not respond to the vaccine and had no detectable VEEV-neutralizing antibody response after vaccination. Likewise, one mouse in the group receiving the

VEEVCOCA _P plasmid did not develop a detectable VEEV-neutralizing antibody response and died after challenge. All mice vaccinated with the VEEVco plasmid developed neutralizing antibodies to VEEV, showed no signs of illness, and survived the challenge. Of note, mice receiving only a single vaccination with 5 μg of the VEEVco plasmid were also completely protected from aerosol VEEV challenge (data not shown).

[97] CELL-MEDIATED IMMUNE RESPONSES OF VACCINATED MICE. Although the most widely accepted correlate of protective immunity against VEEV is antibody responses directed against the envelope glycoproteins (Bennett et al. (2000) Vaccine 19:337-47; Hart et al. (2001) Vaccine 20:616-22; and Phillpotts et al. (2002) Vaccine 20: 1497-504), recent studies have indicated a possible role for certain populations of T-lymphocytes in protection against VEEV in mice. See Brooke et al. (2010) J Virol 84:4556-68; Jones et al. (2003) Virus Res 91 :255-9; Paessler et al. (2007) Virology 367:307-23; and Yun et al. (2009) Vaccine 27:4064-73. Thus, as an indirect measure of the ability of the VEEVco plasmid to elicit cell mediated immune responses, IgG antibody subtypes in pooled sera obtained from mice in the challenge study after their second vaccination was assayed. Mice vaccinated with TC-83 had a preponderance of IgG2a antibodies associated with a predicted Thl skew in immune response (Fig. 6A). In contrast, mice vaccinated with the VEEVco plasmid delivered by i.m. EP had comparable IgGl and IgG2a antibody titers indicative of a more balanced Thl/Th2 immune response.

[98] To directly evaluate the possibility that the VEEVco plasmid could elicit cell- mediated immune responses, groups of female BALB/c mice (N = 6) were vaccinated twice at a 3-week interval 5 μg of empty vector or VEEVco plasmid delivered by i.m. EP. Two weeks after the second vaccination, splenocytes isolated from the vaccinated mice were restimulated with no peptide, Concanavalin A, a β- galactosidase control peptide, or pools of overlapping peptides spanning the VEEV IAB E2 or El envelope glycoproteins and analyzed by IFN-γ ELISPOT. Splenocytes restimulated with Concanavalin A produced a number of spots that were too numerous to count (data not shown). Although splenocytes restimulated with no peptide or with the β-galactosidase peptide failed to produce a response in this assay, those restimulated with pooled peptides representing the VEEV E2 or El protein produced measurable IFN-γ responses (Fig. 6B). Interestingly, the mean IFN-γ response of splenocytes restimulated with the VEEV E2 pooled peptides was significantly higher than that of those restimulated with the VEEV El pooled peptides.

[99] DURABILITY OF THE ANTIBODY RESPONSE TO VEEVco I RABBITS. Although rabbits are not traditionally used as a challenge model for VEEV, their large muscle size permits EP delivery of higher doses of DNA plasmids that are similar to those expected to be delivered to humans. Thus, rabbits are a useful model for studying antibody durability after vaccination. To assess the durability of the antibody response to VEEVco, 5 female New Zealand White rabbits were vaccinated with 500 μg of the VEEVco plasmid delivered by i.m. EP on days 0, 28, and 230, and their antibody responses to VEEV were measured on days 0, 28, 42, 230, 266, and 349 by PR T. The rabbits produced high titers of VEEV-neutralizing antibodies after a single vaccination with the VEEVco DNA construct and upon receiving a second vaccination, their mean PRNTgo titer was significantly boosted (Fig. 7). The PRNTgo titer declined between days 42 and 230, yet remained remarkably high, with a mean titer of 1 : 1 ,000 at day 230. In addition, the mean PRNTgo titer was significantly boosted with an additional DNA vaccination performed on day 230, and the level of VEEV-neutralizing antibodies observed on day 266 was not significantly different from that observed on day 42.

[ 100] IMMUNOGENICITY AND PROTECTIVE EFFICACY OF THE VEEVco PLASMID IN

CYNOMOLGUS MACAQUES. The immunogenicity and protective efficacy of the VEEVco plasmid was assessed using an established model of VEEV infection of nonhuman primates that has been refined by the use of telemetry to evaluate fever responses. See Pratt et al. (1998) Vaccine 16:1056-64. Groups of four adult male cynomolgus macaques were vaccinated by i.m. EP with each animal receiving 500 μg of the empty vector DNA plasmid or 500 μg or 50 μg of the VEEVco plasmid at days 0 and 56. Neutralizing antibodies were detected in all of the macaques after a single vaccination with either dose of the VEEVco plasmid, and a second vaccination significantly boosted the mean PRNTgo titers (Fig. 8). In addition, the mean PRNTgo titers were not significantly different between the 500^g and 50^g dose groups at any of the time points.

[101] On day 112, the macaques were challenged with VEEV IAB, strain Trinidad donkey, administered by the aerosol route, and the average inhaled dose was calculated to be 3 x 10 ⁸ PFU or about 300 median effective doses (ED ₅₀ ). Serum viremia was detected in macaques vaccinated with control empty vector on day 1 , peaked on day 2 with a mean group titer of 1,576 PFU/ml, and persisted for 4 days post-challenge (Fig. 9A). In contrast, the macaques receiving either dose of the VEEVco plasmid were aviremic in all post-challenge serum samples tested.

[102] The protective efficacy of the vaccine was also assessed by telemetry

monitoring of the post-challenge fever responses. Macaques vaccinated with the empty vector DNA developed high biphasic fevers that appeared within 24 hours of VEEV challenge and had peaks at days 2 and 6 post-challenge (Fig. 9B). Macaques receiving either 500 μg or 50 μg of the VEEVco plasmid also developed fevers after VEEV aerosol challenge, but these peaked at day 3 post-challenge and the mean temperature elevations were significantly lower than those of macaques receiving the empty vector DNA at day 1 (p < 0.05), day 2 (p < 0.0001), and day 6 (p < 0.05). In addition, there was no significant difference in the mean temperature elevations of macaques receiving the 500 μg or 50 μg dose of the VEEVco plasmid at any time (p > 0.05).

[103] To monitor lymphopenia, peripheral blood lymphocyte (PBL) counts

determined for post-challenge blood samples collected from all macaques on days 1 to 7 after VEEV challenge were compared to average baseline counts determined for pre-challenge blood samples collected on days -3 to -1. The PBL counts of macaques vaccinated with the empty- vector DNA plasmid dropped an average of 58.6% from baseline within 24 hours of VEEV exposure and these macaques remained

lymphopenic for an average of 6 days post-challenge (Fig. 9C). In contrast, the PBL counts of macaques vaccinated with 500 μg or 50 μg of the VEEVco plasmid dropped an average of 18.5% and 28.1% from baseline, respectively, at 24 hours after challenge and this lymphopenia averaged 3 days for both groups. Over the entire 7 day post-challenge observation period, the macaques vaccinated with the empty- vector DNA developed a lymphopenia that was characterized by an average change of -27.5% from baseline PBL counts, while that of macaques vaccinated with 500 μg or 50 μg of the VEEVco plasmid was -6.4% and 1.1%, respectively.

[104] The macaques were also monitored for clinical signs of disease after aerosol

VEEV challenge. Macaques vaccinated with empty vector DNA displayed mild signs of disease including depression, anorexia, and slightly reduced response to stimuli beginning on day 2 post-challenge, which coordinated with the time of peak serum viremia and the initial peak of fever responses. From post-challenge days 4 to 6, at the time of the secondary peak of fever responses, increased signs of disease including hunched posture with the back turned toward the observer and neurological signs to include loss of coordination and occasional tremors were observed for these macaques. By day 8 post-challenge, and corresponding to a reduction in the severity of the fever responses, only mild signs of disease were evident in these macaques, and on day 9 post-challenge their behavior returned to normal. In contrast, macaques that received either 500 μg or 50 μg of the VEEVco plasmid displayed limited clinical signs of disease with only slight inactivity observed for some macaques on days 3 and 4 post-challenge.

[105] As provided above, administration of the VEEVco plasmid provided

significantly higher ELISA titers at low doses as compared to the VEEVW _T plasmid in mice. Surprisingly, it was found that the VEEVco plasmid not only elicited higher levels of total IgG, but also elicited greater concentrations of neutralizing antibody titers against VEEV. In addition, as provided above, complete protection from lethal aerosol VEEV challenge of mice vaccinated by i.m. EP with VEEVco plasmid was observed. Moreover, a balanced IgGl :IgG2a antibody response to VEEV, which is predictive of a balanced Thl :Th2 immune response, is provided by administration of VEEVco plasmid.

[ 106] MULTIVALENT EEV DNA VACCINE

[ 107] MULTIVALENT EEV DNA VACCINE IN MICE. The present invention also

provides a single vaccine formulation that can provide simultaneous protection against VEEV, EEEV, and WEEV in subjects, such as humans. Owing to the flexibility of the DNA vaccination platform, multivalent vaccines can be created by combination and co-delivery of multiple different plasmids. Previous studies have demonstrated that multivalent DNA vaccine combinations of plasmids expressing genes from diverse pathogens may provide an effective means of vaccination against multiple pathogens. See Riemenschneider et al. (2003) Vaccine 21 :4071-80; and Spik et al. (2006) Vaccine 24:4657-66. However, immune interference has been observed with combinations of plasmids having genes of similar pathogens, e.g. plasmids having genes of substantially similar sequences. See also e.g. McClain et al. (1998) J Infect Dis 177:634-41; and Pittman et al. (2009) Vaccine 27:4879-4882 (examples of known interference with the existing alphavirus IND vaccines).

[108] In an initial study to determine whether a multivalent EEV DNA vaccine could be immunogenic and protective, groups of ten female BALB/c mice were vaccinated by PMED three times at 3 -week intervals with 4 μg of the VEEVW _T plasmid or a WT combination comprising 4 μg each of the VEEVW _T? EEEVW _T , and WEEVW _T plasmids. A negative control group received the plasmid vector with no insert, and a positive control group received TC-83. Serum samples obtained 3 weeks after each vaccination were assayed for total anti-VEEV IgG antibodies by ELISA. The mean ELISA titers of mice vaccinated with the VEEVW _T plasmid alone were higher than those of mice vaccinated with the WT combination after each of the three vaccinations (Fig. 10A). Mice vaccinated with the VEEVW _T plasmid alone or with the WT combination displayed similar low PRNT titers. To assess vaccine efficacy, the mice from all groups were challenged four weeks after the final vaccination with about 10 ⁴ PFU (> 1,000 LD ₅₀ ) of VEEV IAB strain Trinidad donkey administered by the aerosol route. Consistent with results from previous aerosol challenge studies (Dupuy et al. (2009) Vaccine 27:4152-60; and Riemenschneider et al. (2003) Vaccine 21 :4071-80), 80% of the mice receiving the VEEVWT plasmid alone survived the challenge (Fig. 10B). Mice receiving the WT combination had a lower survival rate of 70%. The reduced immunogenicity and protective efficacy of the WT combination compared to the VEEV _W T plasmid alone was not altogether unexpected, as immune interference has been observed after sequential administration of investigational alphavirus vaccines in humans. See McClain et al. (1998) J Infect Dis 177:634-41 ; and Pittman et al. (2009) Vaccine 27:4879-4882.

] To determine whether the immunogenicity and protective efficacy of a multivalent EEV DNA vaccine could be improved, groups of ten female BALB/c mice were vaccinated by i.m. EP twice at a 3-week interval with 5 μg of the VEEVco plasmid or a combination composition comprising 5 μg each of the VEEVco _? EEEVco, and WEEVco plasmids. A negative control group received the plasmid vector with no insert, and a positive control group received TC-83. Serum samples obtained 3 weeks after each vaccination were assayed for total anti-VEEV IgG antibodies by ELISA and for VEEV -neutralizing antibodies by PRNT. The mean ELISA titers of mice vaccinated with the VEEVco plasmid alone were not significantly different from those of mice vaccinated with the combination

composition after each of the three vaccinations (Fig. 1 1 A); however, the mean PRNT go titers elicited with the combination composition were lower than those for the VEEVco plasmid alone after each vaccination (Fig. 1 IB). To assess vaccine efficacy, the mice from all groups were challenged four weeks after the final vaccination with about 10 ⁴ PFU (> 1,000 LD ₅₀ ) of VEEV IAB strain Trinidad donkey administered by the aerosol route. Surprisingly, unlike the WT combination, mice vaccinated with the individual VEEVco plasmid and with the CO combination composition were completely protected from challenge (Fig. 12).

] To evaluate the cell-mediated immune responses, groups of female BALB/c mice (N = 6) were vaccinated twice at a 3 -week interval with 5 μg of empty vector or VEEVco plasmid or a combination composition comprising 5 μg each of the

VEEVco _? EEEVco, and WEEVco plasmids delivered by i.m. EP. Two weeks after the second vaccination, splenocytes isolated from the vaccinated mice were restimulated with no peptide, Concanavalin A, a β-galactosidase control peptide, or pools of overlapping peptides spanning the VEEV IAB E2 or El envelope

glycoproteins and analyzed by IFN-γ ELISPOT. Splenocytes restimulated with Concanavalin A produced a number of spots that were too numerous to count (data not shown). Splenocytes restimulated with no peptide or with the β-galactosidase peptide failed to produce a response in this assay. After restimulation with pooled peptides representing the VEEV E2 or El protein, splenocytes obtained from mice vaccinated with the VEEVco plasmid alone and those vaccinated with the

combination composition produced measurable IFN-γ responses (Fig. 13). However, the mean IFN-γ response was lower for the combination composition. Interestingly, the mean IFN-γ response of splenocytes restimulated with the VEEV E2 pooled peptides was significantly higher than that of those restimulated with the VEEV El pooled peptides for the VEEVco plasmid and combination composition groups.

[I l l] MULTIVALENT EEV DNA VACCINE IN RABBITS. TO assess the

immunogenicity of the multivalent EEV DNA vaccine in another species, female New Zealand White rabbits (N= 5) were vaccinated with 500 μg of the VEEVco plasmid or a combination composition comprising 500 μg each of the VEEVco _? EEEVco, and WEEVco plasmids delivered by i.m. EP on days 0, 28, and 230, and their antibody responses to VEEV were measured on days 0, 28, 42, 230, 266, and 349 by PRNT. The rabbits of both groups produced high titers of VEEV-neutralizing antibodies after a single vaccination and upon receiving a second vaccination, their mean PRNTgo titers were significantly boosted (Fig. 14). The PRNTgo titer of rabbits receiving the combination composition was lower than those of rabbits receiving the individual VEEVco plasmid on all days except day 230, yet they still remained remarkably high. In addition, the mean PRNTgo titer of the combination composition was boosted to a lesser degree after an additional DNA vaccination performed on day 230 as compared to the individual VEEVco plasmid.

[1 12] B. EEEV

[1 13] Experiments similar to those conducted for VEEV nucleic acid molecules and VEEV challenges were also conducted for EEEV sequences and challenges in accordance with the sequences and methods described herein.

[1 14] The sequences of the EEEV nucleic acid molecules are as follows:

[1 15] EEEVco: ATGAGCCTGGCCACCGTGATGTGCGTGCTGGCCAACATCACCT TCCCT TGCGACCAG CCCCCCTGCATGCCCTGCTGCTACGAGAAGAACCCCCACGAGACCCTGACCATGCTG GAACAGAACTACGACAGCCGGGCCTACGACCAGCTGCTGGACGCCGCCGTGAAGTGC AACGCCAGGCGGACCAGGCGGGACCTGGACACCCACT TCACCCAGTACAAGCTGGCC AGGCCCTACATCGCCGACTGCCCCAACTGCGGCCACAGCAGATGCGACAGCCCCATC GCCATCGAGGAAGTGAGAGGCGACGCCCATGCTGGAGTCATCCGGATCCAGACCAGC GCCATGT TCGGCCTGAAAACCGACGGCGTGGACCTGGCCTACATGAGCT TCATGAAC GGCAAGACCCAGAAGAGCATCAAGATCGACAACCTGCACGTGCGGACCTCCGCCCCC TGCAGCCTGGTGTCCCACCACGGCTACTACATCCTGGCCCAGTGCCCCCCTGGCGAC ACCGTGACCGTGGGCT TCCACGACGGCCCCAACCGGCACACCTGCACCGTGGCCCAC AAGGTGGAGT TCCGGCCCGTGGGCCGGGAGAAGTACCGGCACCCCCCCGAGCACGGC GTGGAGCTGCCCTGCAACCGGTACACCCACAAGCGGGCCGACCAGGGCCACTACGTG GAGATGCACCAGCCCGGCCTGGTGGCCGACCACAGCCTGCTGTCCATCCACAGCGCC AAGGTGAAAATCACCGTGCCCAGCGGAGCCCAGGTGAAGTACTACTGCAAGTGCCCC GACGTGCGGGAGGGCATCACCAGCAGCGACCACACCACCACCTGTACCGACGTGAAG CAGTGCAGGGCCTACCTGATCGACAACAAGAAATGGGTGTACAACAGCGGCAGGCTG CCCAGAGGCGAGGGCGACACCT TCAAGGGCAAGCTGCACGTGCCCT TCGTGCCCGTG AAGGCCAAGTGCATCGCCACCCTGGCCCCCGAGCCCCTGGTGGAGCACAAGCACCGG ACCCTGATCCTGCACCTGCACCCCGACCACCCCACCCTGCTGACCACCAGAAGCCTG GGCAGCGACGCCAACCCCACCCGGCAGTGGATCGAGCGGCCCACCACCGTGAACT T T ACCGTGACCGGCGAGGGCCTGGAATACACCTGGGGCAACCACCCCCCCAAGAGAGTG TGGGCCCAGGAAAGCGGCGAGGGCAACCCTCACGGCTGGCCCCACGAAGTGGTGGTC AC AC ACAACAGATACCCCCTGACCACCATCATCGGCCTGTGCACCTGCGTGGCC ATCATCATGGTGTCCTGCGTGACCAGCGTGTGGCTGCTGTGCCGGACCCGGAACCTG TGCATCACCCCCTATAAGCTGGCCCCCAACGCCCAGGTGCCCATCCTGCTGGCCCTG CTGTGCTGCATCAAGCCCACCAGGGCCGACGACACCCTGCAGGTGCTGAACTACCTG TGGAACAACAACCAGAACT TCT TCTGGATGCAGACACTGATCCCCCTGGCCGCCCTG ATCGTGTGCATGCGGATGCTGCGGTGCCTGT TCTGCTGCGGCCCTGCCT TCCTGCTG GTGTGCGGAGCCCTGGGCGCCGCCGCCTACGAGCACACCGCCGTGATGCCCAACAAA GTGGGCATCCCCTACAAGGCCCTGGTGGAAAGGCCCGGCTACGCCCCCGTGCACCTG CAGATCCAGCTGGTGAACACCCGGATCATCCCCAGCACCAATCTGGAATACATCACC TGCAAGTACAAGACCAAGGTGCCCAGCCCCGTGGTGAAGTGCTGCGGCGCCACCCAG TGCACCAGCAAGCCCCACCCCGACTACCAGTGCCAGGTGT TCACCGGCGTGTACCCC T TCATGTGGGGCGGAGCCTACTGCT TCTGCGACACCGAGAACACCCAGATGAGCGAG GCCTACGTGGAGCGGAGCGAGGAATGCAGCATCGACCACGCCAAGGCCTACAAGGTG CACACCGGCACAGTGCAGGCCATGGTGAACATCACCTACGGCAGCGTGAGCTGGCGG AGCGCCGACGTGTACGTGAATGGCGAGACCCCCGCCAAGATCGGCGACGCCAAGCTG ATCATCGGCCCCCTGAGCAGCGCCTGGTCCCCCT TCGACAACAAAGTGGTGGTGTAT GGCCACGAGGTGTACAACTACGACT TCCCCGAGTACGGCACCGGCAAGGCCGGCAGC T TCGGCGACCTGCAGAGCCGGACCAGCACCAGCAACGACCTGTACGCCAACACCAAC CTGAAGCTGCAGCGGCCCCAGGCCGGCATCGTGCACACCCCT T TCACCCAGGCCCCC AGCGGCT TCGAGCGGTGGAAGCGGGACAAAGGCGCCCCTCTGAACGACGTGGCCCCC T TCGGCTGCAGCATCGCCCTGGAACCCCTGCGGGCCGAGAACTGCGCCGTGGGCAGC ATCCCCATCAGCATCGACATCCCCGACGCCGCCT TCACCAGGATCTCCGAGACCCCC ACCGTGAGCGACCTGGAATGCAAGATCACCGAGTGCACCTACGCCAGCGACT TCGGC GGCATCGCCACAGTGGCCTACAAGTCCAGCAAGGCCGGAAACTGCCCCATCCACTCC CCCTCCGGCGTGGCCGTGATCAAAGAAAACGACGTGACCCTGGCCGAGAGCGGCAGC T TCACCT TCCACT TCAGCACCGCCAACATCCACCCCGCCT TCAAGCTGCAGGTGTGC ACCAGCGCCGTGACCTGCAAGGGCGACTGCAAGCCCCCCAAGGACCACATCGTGGAC TACCCCGCCCAGCACACCGAGAGCT TCACCTCCGCCATCAGCGCCACCGCCTGGTCC TGGCTGAAGGTGCTGGTCGGCGGCACCTCCGCCT TCATCGTGCTGGGCCTGATCGCC ACAGCCGTGGTGGCCCTGGTGCTGT TCT TCCACCGGCACTGATGA (SEQ ID N0:3). As used herein, a plasmid containing SEQ ID NO:3 is referred to as "EEEVco plasmid".

EEEVCOCAP:

ATGTTCCCTTACCCCACCCTGAACTACCCCCCCATGGCCCCCATCAACCCCATGGCC TACCGGGACCCTAACCCTCCCAGACGCCGGTGGCGGCCCTTCAGACCCCCTCTGGCC GCCCAGATCGAGGACCTGCGGCGGAGCATTGCCAGCCTGACCCTGAAGCAGAGAGCC CCCAACCCTCCTGCCGGCCCTCCCGCCAAGCGGAAGAAGCCTGCCCCCAAGCCCAAG CCCGCCCAGGCCAAGAAGAAGAGGCCCCCTCCCCCTGCCAAGAAGCAGAAGCGGAAG CCTAAGCCCGGCAAGCGGCAGCGGATGTGCATGAAGCTGGAAAGCGACAAGACCTTC CCCATCATGCTGAACGGCCAGGTGAACGGCTACGCCTGCGTGGTGGGGGGCAGAGTG TTCAAGCCCCTGCACGTGGAGGGCCGGATCGACAACGAGCAGCTGGCCGCCATCAAG CTGAAGAAGGCCAGCATCTACGACCTGGAATACGGCGACGTGCCCCAGTGCATGAAG TCCGACACCCTGCAGTACACCAGCGACAAGCCCCCTGGCTTCTACAACTGGCACCAC GGGGCCGTGCAGTACGAGAACAACAGATTCACCGTGCCCAGAGGCGTGGGCGGCAAG GGCGACAGCGGCAGACCCATCCTGGACAACAAGGGCCGGGTGGTGGCCATCGTGCTG GGCGGCGTGAACGAGGGCAGCCGGACCGCCCTGAGCGTGGTGACCTGGAACCAGAAA GGCGTGACCGTGAAGGACACCCCCGAGGGCAGCGAGCCTTGGAGCCTGGCCACCGTG ATGTGCGTGCTGGCCAACATCACCTTCCCTTGCGACCAGCCCCCCTGCATGCCCTGC TGCTACGAGAAGAACCCCCACGAGACCCTGACCATGCTGGAACAGAACTACGACAGC CGGGCCTACGACCAGCTGCTGGACGCCGCCGTGAAGTGCAACGCCAGGCGGACCAGG CGGGACCTGGACACCCACTTCACCCAGTACAAGCTGGCCAGGCCCTACATCGCCGAC TGCCCCAACTGCGGCCACAGCAGATGCGACAGCCCCATCGCCATCGAGGAAGTGAGA GGCGACGCCCATGCTGGAGTCATCCGGATCCAGACCAGCGCCATGTTCGGCCTGAAA ACCGACGGCGTGGACCTGGCCTACATGAGCTTCATGAACGGCAAGACCCAGAAGAGC ATCAAGATCGACAACCTGCACGTGCGGACCTCCGCCCCCTGCAGCCTGGTGTCCCAC CACGGCTACTACATCCTGGCCCAGTGCCCCCCTGGCGACACCGTGACCGTGGGCTTC CACGACGGCCCCAACCGGCACACCTGCACCGTGGCCCACAAGGTGGAGTTCCGGCCC GTGGGCCGGGAGAAGTACCGGCACCCCCCCGAGCACGGCGTGGAGCTGCCCTGCAAC CGGTACACCCACAAGCGGGCCGACCAGGGCCACTACGTGGAGATGCACCAGCCCGGC CTGGTGGCCGACCACAGCCTGCTGTCCATCCACAGCGCCAAGGTGAAAATCACCGTG CCCAGCGGAGCCCAGGTGAAGTACTACTGCAAGTGCCCCGACGTGCGGGAGGGCATC ACCAGCAGCGACCACACCACCACCTGTACCGACGTGAAGCAGTGCAGGGCCTACCTG ATCGACAACAAGAAATGGGTGTACAACAGCGGCAGGCTGCCCAGAGGCGAGGGCGAC ACCTTCAAGGGCAAGCTGCACGTGCCCTTCGTGCCCGTGAAGGCCAAGTGCATCGCC ACCCTGGCCCCCGAGCCCCTGGTGGAGCACAAGCACCGGACCCTGATCCTGCACCTG CACCCCGACCACCCCACCCTGCTGACCACCAGAAGCCTGGGCAGCGACGCCAACCCC ACCCGGCAGTGGATCGAGCGGCCCACCACCGTGAACTTTACCGTGACCGGCGAGGGC CTGGAATACACCTGGGGCAACCACCCCCCCAAGAGAGTGTGGGCCCAGGAAAGCGGC GAGGGCAACCCTCACGGCTGGCCCCACGAAGTGGTGGTCTACTACTACAACAGATAC CCCCTGACCACCATCATCGGCCTGTGCACCTGCGTGGCCATCATCATGGTGTCCTGC GTGACCAGCGTGTGGCTGCTGTGCCGGACCCGGAACCTGTGCATCACCCCCTATAAG CTGGCCCCCAACGCCCAGGTGCCCATCCTGCTGGCCCTGCTGTGCTGCATCAAGCCC ACCAGGGCCGACGACACCCTGCAGGTGCTGAACTACCTGTGGAACAACAACCAGAAC TTCTTCTGGATGCAGACACTGATCCCCCTGGCCGCCCTGATCGTGTGCATGCGGATG CTGCGGTGCCTGTTCTGCTGCGGCCCTGCCTTCCTGCTGGTGTGCGGAGCCCTGGGC GCCGCCGCCTACGAGCACACCGCCGTGATGCCCAACAAAGTGGGCATCCCCTACAAG GCCCTGGTGGAAAGGCCCGGCTACGCCCCCGTGCACCTGCAGATCCAGCTGGTGAAC ACCCGGATCATCCCCAGCACCAATCTGGAATACATCACCTGCAAGTACAAGACCAAG GTGCCCAGCCCCGTGGTGAAGTGCTGCGGCGCCACCCAGTGCACCAGCAAGCCCCAC CCCGACTACCAGTGCCAGGTGTTCACCGGCGTGTACCCCTTCATGTGGGGCGGAGCC TACTGCT TCTGCGACACCGAGAACACCCAGATGAGCGAGGCCTACGTGGAGCGGAGC

GAGGAATGCAGCATCGACCACGCCAAGGCCTACAAGGTGCACACCGGCACAGTGCAG

GCCATGGTGAACATCACCTACGGCAGCGTGAGCTGGCGGAGCGCCGACGTGTACGTG

AATGGCGAGACCCCCGCCAAGATCGGCGACGCCAAGCTGATCATCGGCCCCCTGAGC

AGCGCCTGGTCCCCCT TCGACAACAAAGTGGTGGTGTATGGCCACGAGGTGTACAAC

TACGACT TCCCCGAGTACGGCACCGGCAAGGCCGGCAGCT TCGGCGACCTGCAGAGC

CGGACCAGCACCAGCAACGACCTGTACGCCAACACCAACCTGAAGCTGCAGCGGCCC

CAGGCCGGCATCGTGCACACCCCT T TCACCCAGGCCCCCAGCGGCT TCGAGCGGTGG

AAGCGGGACAAAGGCGCCCCTCTGAACGACGTGGCCCCCT TCGGCTGCAGCATCGCC

CTGGAACCCCTGCGGGCCGAGAACTGCGCCGTGGGCAGCATCCCCATCAGCATCGAC

ATCCCCGACGCCGCCT TCACCAGGATCTCCGAGACCCCCACCGTGAGCGACCTGGAA

TGCAAGATCACCGAGTGCACCTACGCCAGCGACT TCGGCGGCATCGCCACAGTGGCC

TACAAGTCCAGCAAGGCCGGAAACTGCCCCATCCACTCCCCCTCCGGCGTGGCCGTG

ATCAAAGAAAACGACGTGACCCTGGCCGAGAGCGGCAGCT TCACCT TCCACT TCAGC

ACCGCCAACATCCACCCCGCCT TCAAGCTGCAGGTGTGCACCAGCGCCGTGACCTGC

AAGGGCGACTGCAAGCCCCCCAAGGACCACATCGTGGACTACCCCGCCCAGCACACC

GAGAGCT TCACCTCCGCCATCAGCGCCACCGCCTGGTCCTGGCTGAAGGTGCTGGTC

GGCGGCACCTCCGCCT TCATCGTGCTGGGCCTGATCGCCACAGCCGTGGTGGCCCTG

GTGCTGT TCT TCCACCGGCACTGATGA (SEQ ID N0:4). As used herein, a plasmid containing SEQ ID NO:4 is referred to as "EEEVCOCA _P plasmid".

EEEV DNA VACCINE IN MICE. An initial study was performed to assess the immunogenicity and protective efficacy of EEEVW _T , a DNA plasmid expressing the wild-type structural proteins (C-E3-E2-6K-E1) of EEEV (strain PE6), both alone and in combination with the VEEVW _T and WEEVW _T plasmids (WT combination). For this, groups of ten female BALB/c mice were vaccinated by PMED three times at 3- week intervals with 4 μg of the EEEVW _T plasmid or the WT combination comprising 4 μg each of the EEEVW _T? VEEV _WT , and WEEV _WT plasmids. A negative control group received 4 μg of the plasmid vector with no insert three times at 3 -week intervals by PMED. A positive control group was vaccinated three times at 3 -week intervals with 0.5 ml of the formalin-inactivated EEEV IND vaccine delivered by subcutaneous injection. Serum samples obtained 3 weeks after each vaccination were assayed for total anti-EEEV IgG antibodies by ELISA, and serum samples obtained three weeks after the third and final vaccination were also assayed for EEEV- neutralizing antibodies by PRNT. The mean ELISA titers of mice vaccinated with the EEEVW _T plasmid alone were higher than those of mice vaccinated with the WT combination after each of the three vaccinations (Fig. 15 A). Mice vaccinated with the EEEVW _T plasmid alone or in the WT combination displayed similar low PRNT titers. To assess vaccine efficacy, the mice from all groups were challenged four weeks after the final vaccination with about 10 ⁴ PFU (-300 LD ₅₀ ) of EEEV strain PE6 administered by the aerosol route. None of the mice vaccinated with EEEVW _T survived the challenge; however, there was a significant delay in the day to death as compared to the negative control mice. Although there was also a complete lack of survival of mice vaccinated with the WT combination, these mice died with similar kinetics as the negative controls. Mice vaccinated with the EEEV IND were protected at a level of 40% (Fig. 15B).

[118] To determine whether the EEEV DNA vaccine could be improved by

employing the same strategy that proved successful for the VEEV DNA vaccine, the wild type 26S structural genes minus the capsid sequence (E3-E2-6K-E1) of EEEV strain FL91-4679 (Genbank accession number AY705241) were codon optimized by subjecting these genes to a GeneOptimizer™ bioinformatic algorithm (Geneart, Regensburg, Germany) for mammalian expression, and the codon optimized genes were synthesized using methods known in the art. The EEEVco plasmid was constructed by cloning these synthesized codon optimized genes into the Notl and BgUl restriction sites of pWRG7077 using methods known in the art. EEEV strain FL91-4679 was chosen because it was unexpectedly found to represent a consensus sequence after performing an alignment of the structural gene sequences of many EEEV strains.

[119] To determine if the EEEVco construct showed improved expression of the envelope glycoproteins as compared to the previous EEEV _WT construct, the proteins expressed in transiently transfected mammalian cells were detected by immune precipitation assay. The expression of the E2 and El envelope glycoproteins from the EEEVco construct was increased approximately tenfold relative to that of the

EEEV _T construct in this assay (Fig. 16A). To further quantify the relative in vitro expression levels of these constructs, the percent of cells positive for E2 and El expression after transient transfection of 5-500 ng of each DNA vaccine plasmid was determined by flow cytometry. A significant increase in the mean percent of cells positive for E2 and El expression was observed for the EEEVco DNA plasmid at the 50-500 ng sample concentrations (Fig. 16B).

[120] In an initial study to determine whether the immunogenicity of the EEEVco plasmid was improved relative to the EEEVW _T plasmid, groups of six female BALB/c mice were vaccinated by i.m. EP three times at 3-week intervals with 25 μg, 5 μg, or 1 μg of DNA. A negative control group received 25 μg of the plasmid vector with no insert. Serum samples obtained 3 weeks after each vaccination were assayed for total anti-EEEV IgG antibodies by ELISA and for EEEV-neutralizing antibodies by PRNT. The mean ELISA titers of mice vaccinated with the EEEVco plasmid were

significantly higher than those of mice vaccinated with the EEEV T construct for the 5 μg and 1 μg dose groups after each of the three vaccinations (Fig. 17A). The mean PRNTgo titer elicited by mice vaccinated with the EEEVco plasmid was significantly higher than that observed for mice vaccinated with the EEEVWT construct for all dose groups at all days (Fig. 17B).

[121] To determine the protective efficacy of the EEEVco DNA vaccine both alone and in the multivalent EEV combination (combination composition), groups of ten female BALB/c mice were vaccinated by i.m. EP three times at 3-week intervals with 5 μg of the EEEVco plasmid or a combination composition comprising 5 μg each of the EEEVco, VEEVco, and WEEVco plasmids. A negative control group received the plasmid vector with no insert, and a positive control group received the formalin- inactivated EEEV IND vaccine. Serum samples obtained 3 weeks after each vaccination were assayed for total anti-EEEV IgG antibodies by ELISA and for EEEV-neutralizing antibodies by PRNT. The mean ELISA titers of mice vaccinated with the EEEVco plasmid alone were not significantly different from those of mice vaccinated with the combination composition after each of the three vaccinations (Fig. 18A). In addition, the mean ELISA titers of mice receiving the individual EEEVco plasmid or the combination composition were higher than that of the EEEV IND after each of the three vaccinations. The mean PRNTgo titers elicited with the EEEVco plasmid alone were higher than those for the combination composition or the EEEV IND vaccine after each vaccination, while those of the combination composition were similar to those of the EEEV IND after each of the three vaccinations (Fig. 18B). To assess vaccine efficacy, the mice from all groups were challenged four weeks after the final vaccination with about 10 ⁴ PFU (-300 LD ₅₀ ) of EEEV strain FL91-4679 administered by the aerosol route. Surprisingly, mice vaccinated with the individual EEEVco plasmid and with the combination composition were completely protected from challenge (Fig. 18). Similar to the results from our previous EEEVWT mouse challenge study, 40% of mice receiving the EEEV IND vaccine survived.

[122] EEEV DNA VACCINE IN RABBITS. TO assess the immunogenicity of the

EEEVco DNA vaccine alone and in the multivalent EEV combination in another species, female New Zealand White rabbits (N = 5) were vaccinated with 500 μg of the EEEVco plasmid or a combination composition comprising 500 μg each of the EEEVco _? VEEVco, and WEEVco plasmids delivered by i.m. EP on days 0, 28, and 230, and their antibody responses to EEEV were measured on days 0, 28, 42, 230, 266, and 349 by PRNT. The rabbits of both groups produced high titers of EEEV- neutralizing antibodies after a single vaccination and upon receiving a second vaccination, their mean PRNTgo titers were significantly boosted (Fig. 20). The PRNT go titer of rabbits receiving the combination composition was lower than those of rabbits receiving the individual VEEVco plasmid on all days, yet they still remained remarkably high. In addition, the mean PRNTgo titers of both groups were boosted after an additional DNA vaccination performed on day 230.

[123] EEEV DNA VACCINE IN CYNOMOLGUS MACAQUES . The immunogenicity and protective efficacy of the EEEVco plasmid, both alone and in the multivalent EEV combination (combination composition), was assessed using an established model of EEEV infection of nonhuman primates. See Reed et al. (2007) J Infect Dis 196:441- 50. Groups of four adult male cynomolgus macaques were vaccinated three times at 4-week intervals with each animal receiving 500 μg of the empty vector DNA plasmid, 500 μg of the EEEVco plasmid, or a combination composition comprising 500 μg each of the EEEVco, VEEVco, and WEEVco plasmid delivered by i.m. EP. Serum samples collected 4 weeks after each vaccination were analyzed for EEEV- neutralizing antibodies by PRNT. Similar mean neutralizing antibody titers were elicited in macaques after a single vaccination with the individual EEEVco plasmid and the combination composition, and the titers of both groups were significantly and similarly boosted after the second vaccination (Fig. 21). The mean titer of neither group was boosted with a third vaccination, and the titers of macaques receiving the EEEVco plasmid alone were significantly higher than those receiving the combination composition at day 85.

[124] Eight weeks after the third and final vaccination, the macaques were

challenged with EEEV strain FL91-4679 administered by the aerosol route, and the average inhaled dose was calculated to be 1.6 x 10 ⁸ PFU. While serum viremia was detected in postchallenge samples from 3 of 4 control macaques vaccinated with empty vector plasmid, no serum viremia was detected in any of the postchallenge samples from macaques receiving either the individual EEEVco plasmid or the combination composition (Fig. 22A). After aerosol EEEV challenge, blinded observations to monitor the macaques for clinical signs of disease, including neurological signs and changes in activity, behavior, and response to stimuli, were performed using established criteria. Macaques vaccinated with empty vector DNA displayed signs of disease beginning on day 2 postchallenge. On day 5 postchallenge, 3 of 4 of these control macaques displayed signs of severe encephalitis and complete inactivity and, having met the established clinical score for a humane endpoint, were euthanized. On day 7 postchallenge, the remaining control macaque also succumbed to disease, met the established clinical score for a humane endpoint, and was euthanized. All of the macaques vaccinated with the individual EEEVco plasmid or with the combination composition survived the challenge and never met the established clinical score for a humane endpoint. However, 2 of 4 of those receiving the individual EEEVco plasmid did display signs of illness and had increased clinical scores after challenge resulting in increased mean clinical scores for this group (Fig. 22B). Surprisingly, those macaques vaccinated with the combination composition did not display signs of illness after challenge. This provides the first indication that a multivalent DNA vaccine comprising VEEVco, EEEVco, and WEEVco plasmids can act synergistically to improve protection from encephalitic alphavirus challenge as compared to the individual DNA vaccines.

[125] C. WEEV

[126] Experiments similar to those conducted for VEEV nucleic acid molecules and

VEEV challenges were also conducted for WEEV sequences and challenges in accordance with the sequence and methods described herein.

[127] The sequences of the WEEV nucleic acid molecules are as follows:

[128] WEEVco:

ATGAGCCTGGTGACCGCCCTGTGCGTGCTGTCCAACGTGACCT TCCCCTGCGACAAG CCCCCCGTGTGCTACAGCCTGGCCCCCGAGCGGACCCTGGACGTGCTGGAAGAGAAC GTGGACAACCCCAACTACGACACCCTGCTGGAAAACGTGCTGAAGTGCCCCAGCAGG CGGCCCAAGCGGAGCATCACCGACGACT TCACCCTGACCAGCCCCTACCTGGGCT TC TGCCCCTACTGCCGGCACAGCGCCCCCTGCT TCAGCCCCATCAAGATCGAGAACGTG TGGGACGAGAGCGACGACGGCAGCATCCGGATCCAGGTGTCCGCCCAGT TCGGCTAC AACCAGGCCGGCACCGCCGACGTGACCAAGT TCCGGTACATGAGCTACGACCACGAC CACGATATCAAGGAAGATAGCATGGAAAAGCTGGCCATCAGCACCAGCGGCCCCTGC AGACGGCTGGGCCACAAGGGCTACT T TCTGCTGGCCCAGTGCCCCCCTGGCGACAGC GTGACCGTGAGCATCACCAGCGGCGCCAGCGAGAACAGCTGCACCGTGGAGAAGAAG ATCCGGCGGAAGT TCGTGGGCCGGGAGGAATACCTGT TCCCCCCCGTGCACGGCAAG CTGGTGAAGTGCCACGTGTACGACCACCTGAAAGAGACCAGCGCCGGCTACATCACC ATGCACCGGCCAGGCCCCCACGCCTACAAGAGCTACCTGGAAGAGGCCAGCGGCGAG GTGTACATCAAGCCCCCCAGCGGCAAGAACGTGACCTACGAGTGCAAGTGCGGCGAC TACAGCACCGGCATCGTGAGCACCCGGACCAAGATGAACGGCTGCACCAAGGCCAAG CAGTGCATCGCCTACAAGCGGGACCAGACCAAGTGGGTGT TCAACAGCCCCGACCTG ATCCGGCACACCGACCACAGCGTGCAGGGCAAACTGCACATCCCCT TCCGGCTGACC CCCACCGTGTGCCCCGTGCCCCTGGCCCACACCCCTACCGTGACAAAGTGGT TCAAG GGCATCACACTGCACCTGACCGCCACCCGGCCCACCCTGCTGACCACCCGGAAGCTG GGCCTGAGGGCCGATGCCACCGCCGAGTGGATCACCGGCACCACCTCCCGGAACT TC AGCGTGGGCAGAGAGGGCCTGGAATACGTCTGGGGCAACCACGAGCCCGTGAGAGTG TGGGCCCAGGAAAGCGCCCCAGGCGACCCCCACGGCTGGCCCCACGAGATCATCATC CACTACTACCACCGGCACCCCGTGTACACCGTGATCGTGCTGTGCGGCGTGGCCCTG GCCATCCTGGTGGGCACCGCCAGCAGCGCCGCCTGCATCGCCAAGGCCAGGCGGGAC TGCCTGACCCCCTACGCCCTGGCCCCCAACGCCACCGTGCCAACCGCCCTGGCCGTG CTGTGCTGCATCCGGCCCACCAACGCCGAGACCT TCGGCGAGACCCTGAACCACCTG TGGT TCAACAACCAGCCCT TCCTGTGGGCCCAGCTGTGCATCCCCCTGGCCGCCCTG ATCATCCTGT TCCGGTGCT TCAGCTGCTGCATGCCT T T TCTGCTGGTCGCCGGCGTG TGCCTGGGCAAGGTGGACGCCT TCGAGCACGCCACCACCGTGCCCAACGTGCCCGGC ATCCCCTACAAGGCCCTGGTGGAGAGGGCCGGCTACGCCCCCCTGAACCTGGAAATC ACCGTGGTGTCCAGCGAGCTGACCCCCTCCACCAACAAAGAATACGTGACCTGCAAG T TCCACACCGTGGTGCCCTCCCCCCAGGTGAAGTGCTGCGGCAGCCTGGAATGCAAG GCCAGCAGCAAGGCCGACTACACCTGCCGGGTGT TCGGCGGCGTGTACCCCT TCATG TGGGGCGGAGCACAGTGCT TCTGCGACTCCGAGAACACCCAGCTGTCCGAGGCCTAC GTGGAGT TCGCCCCCGACTGCACCATCGACCACGCCGTGGCCCTGAAGGTGCACACA GCCGCCCTGAAAGTGGGCCTGCGGATCGTGTACGGCAACACCACCGCCAGGCTGGAC ACCT TCGTGAACGGCGTGACCCCCGGCAGCAGCCGGGACCTGAAGGTGATCGCCGGA CCCATCTCCGCCGCCT TCAGCCCCT TCGACCACAAGGTGGTGATCCGGAAGGGCCTG GTGTACAACTACGACT TCCCCGAGTACGGCGCCATGAACCCTGGCGCCT TCGGCGAC ATCCAGGCCAGCTCCCTGGACGCCACCGACATCGTGGCCCGGACCGACATCCGGCTG CTGAAGCCCAGCGTGAAGAACATCCACGTGCCCTACACCCAGGCCGTGAGCGGCTAC GAGATGTGGAAGAACAACAGCGGCAGACCCCTGCAGGAAACCGCCCCCT TCGGCTGC AAGATCGAGGTGGAGCCCCTGCGGGCCACCAACTGCGCCTACGGCCACATCCCCATC AGCATCGACATCCCCGACGCCGCCT TCGTGCGGAGCAGCGAGAGCCCCACCATCCTG GAAGTGAGCTGTACCGTGGCCGACTGCATCTACAGCGCCGACT TCGGCGGCTCCCTG ACCCTGCAGTACAAGGCCAACCGGGAGGGCCACTGCCCCGTGCACAGCCACAGCACC ACCGCCGTGCTGAAAGAGGCCACCACCCACGTCACCGCCACAGGCAGCATCACCCTG CACT TCAGCACCAGCTCCCCCCAGGCCAACT TCATCGTGAGCCTGTGCGGCAAGAAA ACCACCTGCAACGCCGAGTGCAAGCCCCCTGCCGACCACATCATCGGCGAGCCTCAC AAGGTGGACCAGGAAT TCCAGGCCGCCGTCAGCAAGACCAGCTGGAACTGGCTGCTG GCCCTGT TCGGCGGAGCCAGCAGCCTGATCGTGGTGGGCCTGAT TGTGCTGGTGTGC AGCAGCATGCTGATCAACACCCGGCGGTGATGA (SEQ ID N0:5). As used herein, a plasmid containing SEQ ID NO: 5 is referred to as "WEEVco plasmid". WEEVCOCAP:

ATGT TCCCCTACCCCCAGCTGAACT TCCCCCCCGTGTACCCCACCAACCCCATGGCC TACCGGGACCCTAATCCACCTAGACGCCGGTGGCGGCCCT TCAGACCCCCTCTGGCC GCCCAGATCGAGGACCTGCGGCGGAGCAT TGCCAACCTGACCT TCAAGCAGAGAGCC CCCAACCCTCCTCCAGGACCCCCTCCCAAGAAGAAGAAGTCCGCCCCCAAGCCCAAG CCCACCCAGCCTAAGAAGAAAAAGCAGCAGGCCAAGAAAACCAAGCGGAAGCCTAAG CCCGGCAAGCGGCAGCGGATGTGCATGAAGCTGGAAAGCGACAAGACCT TCCCCATC ATGCTGAACGGCCAGGTGAACGGCTACGCCTGCGTGGTGGGCGGGAGACTGATGAAG CCCCTGCACGTGGAGGGCAAGATCGACAACGAGCAGCTGGCCGCCGTGAAGCTGAAG AAAGCCAGCATGTACGACCTGGAATACGGCGACGTGCCCCAGAACATGAAGTCCGAC ACCCTGCAGTACACCAGCGACAAGCCCCCTGGCT TCTACAACTGGCACCATGGCGCC GTGCAGTACGAGAACGGCCGGT TCACCGTGCCCAGAGGCGTGGGCGGCAAGGGCGAC AGCGGCAGACCCATCCTGGACAACCGGGGCAGAGTGGTGGCCATCGTGCTGGGCGGA GCCAACGAGGGCACCCGGACAGCCCTGAGCGTGGTGACCTGGAACCAGAAAGGCGTG ACCATCAAGGACACCCCCGAGGGCAGCGAGCCCTGGAGCCTGGTGACCGCCCTGTGC GTGCTGTCCAACGTGACCT TCCCCTGCGACAAGCCCCCCGTGTGCTACAGCCTGGCC CCCGAGCGGACCCTGGACGTGCTGGAAGAGAACGTGGACAACCCCAACTACGACACC CTGCTGGAAAACGTGCTGAAGTGCCCCAGCAGGCGGCCCAAGCGGAGCATCACCGAC GACT TCACCCTGACCAGCCCCTACCTGGGCT TCTGCCCCTACTGCCGGCACAGCGCC CCCTGCT TCAGCCCCATCAAGATCGAGAACGTGTGGGACGAGAGCGACGACGGCAGC ATCCGGATCCAGGTGTCCGCCCAGT TCGGCTACAACCAGGCCGGCACCGCCGACGTG ACCAAGT TCCGGTACATGAGCTACGACCACGACCACGATATCAAGGAAGATAGCATG GAAAAGCTGGCCATCAGCACCAGCGGCCCCTGCAGACGGCTGGGCCACAAGGGCTAC T T TCTGCTGGCCCAGTGCCCCCCTGGCGACAGCGTGACCGTGAGCATCACCAGCGGC GCCAGCGAGAACAGCTGCACCGTGGAGAAGAAGATCCGGCGGAAGT TCGTGGGCCGG GAGGAATACCTGT TCCCCCCCGTGCACGGCAAGCTGGTGAAGTGCCACGTGTACGAC CACCTGAAAGAGACCAGCGCCGGCTACATCACCATGCACCGGCCAGGCCCCCACGCC TACAAGAGCTACCTGGAAGAGGCCAGCGGCGAGGTGTACATCAAGCCCCCCAGCGGC AAGAACGTGACCTACGAGTGCAAGTGCGGCGACTACAGCACCGGCATCGTGAGCACC CGGACCAAGATGAACGGCTGCACCAAGGCCAAGCAGTGCATCGCCTACAAGCGGGAC CAGACCAAGTGGGTGT TCAACAGCCCCGACCTGATCCGGCACACCGACCACAGCGTG CAGGGCAAACTGCACATCCCCT TCCGGCTGACCCCCACCGTGTGCCCCGTGCCCCTG GCCCACACCCCTACCGTGACAAAGTGGT TCAAGGGCATCACACTGCACCTGACCGCC ACCCGGCCCACCCTGCTGACCACCCGGAAGCTGGGCCTGAGGGCCGATGCCACCGCC GAGTGGATCACCGGCACCACCTCCCGGAACT TCAGCGTGGGCAGAGAGGGCCTGGAA TACGTCTGGGGCAACCACGAGCCCGTGAGAGTGTGGGCCCAGGAAAGCGCCCCAGGC GACCCCCACGGCTGGCCCCACGAGATCATCATCCACTACTACCACCGGCACCCCGTG TACACCGTGATCGTGCTGTGCGGCGTGGCCCTGGCCATCCTGGTGGGCACCGCCAGC AGCGCCGCCTGCATCGCCAAGGCCAGGCGGGACTGCCTGACCCCCTACGCCCTGGCC CCCAACGCCACCGTGCCAACCGCCCTGGCCGTGCTGTGCTGCATCCGGCCCACCAAC GCCGAGACCT TCGGCGAGACCCTGAACCACCTGTGGT TCAACAACCAGCCCT TCCTG TGGGCCCAGCTGTGCATCCCCCTGGCCGCCCTGATCATCCTGT TCCGGTGCT TCAGC TGCTGCATGCCT T T TCTGCTGGTCGCCGGCGTGTGCCTGGGCAAGGTGGACGCCT TC GAGCACGCCACCACCGTGCCCAACGTGCCCGGCATCCCCTACAAGGCCCTGGTGGAG AGGGCCGGCTACGCCCCCCTGAACCTGGAAATCACCGTGGTGTCCAGCGAGCTGACC CCCTCCACCAACAAAGAATACGTGACCTGCAAGT TCCACACCGTGGTGCCCTCCCCC CAGGTGAAGTGCTGCGGCAGCCTGGAATGCAAGGCCAGCAGCAAGGCCGACTACACC TGCCGGGTGT TCGGCGGCGTGTACCCCT TCATGTGGGGCGGAGCACAGTGCT TCTGC GACTCCGAGAACACCCAGCTGTCCGAGGCCTACGTGGAGT TCGCCCCCGACTGCACC ATCGACCACGCCGTGGCCCTGAAGGTGCACACAGCCGCCCTGAAAGTGGGCCTGCGG ATCGTGTACGGCAACACCACCGCCAGGCTGGACACCT TCGTGAACGGCGTGACCCCC GGCAGCAGCCGGGACCTGAAGGTGATCGCCGGACCCATCTCCGCCGCCT TCAGCCCC T TCGACCACAAGGTGGTGATCCGGAAGGGCCTGGTGTACAACTACGACT TCCCCGAG TACGGCGCCATGAACCCTGGCGCCT TCGGCGACATCCAGGCCAGCTCCCTGGACGCC ACCGACATCGTGGCCCGGACCGACATCCGGCTGCTGAAGCCCAGCGTGAAGAACATC CACGTGCCCTACACCCAGGCCGTGAGCGGCTACGAGATGTGGAAGAACAACAGCGGC AGACCCCTGCAGGAAACCGCCCCCT TCGGCTGCAAGATCGAGGTGGAGCCCCTGCGG GCCACCAACTGCGCCTACGGCCACATCCCCATCAGCATCGACATCCCCGACGCCGCC T TCGTGCGGAGCAGCGAGAGCCCCACCATCCTGGAAGTGAGCTGTACCGTGGCCGAC TGCATCTACAGCGCCGACT TCGGCGGCTCCCTGACCCTGCAGTACAAGGCCAACCGG GAGGGCCACTGCCCCGTGCACAGCCACAGCACCACCGCCGTGCTGAAAGAGGCCACC ACCCACGTCACCGCCACAGGCAGCATCACCCTGCACT TCAGCACCAGCTCCCCCCAG GCCAACT TCATCGTGAGCCTGTGCGGCAAGAAAACCACCTGCAACGCCGAGTGCAAG CCCCCTGCCGACCACATCATCGGCGAGCCTCACAAGGTGGACCAGGAAT TCCAGGCC GCCGTCAGCAAGACCAGCTGGAACTGGCTGCTGGCCCTGT TCGGCGGAGCCAGCAGC CTGATCGTGGTGGGCCTGAT TGTGCTGGTGTGCAGCAGCATGCTGATCAACACCCGG CGGTGATGA (SEQ ID NO:6). As used herein, a plasmid containing SEQ ID NO: 6 is referred to as "WEEVCOCAP plasmid".

[130] WEEV DNA VACCINE IN MICE. An initial study was performed to assess the immunogenicity and protective efficacy of WEEVW _T , a DNA plasmid expressing the wild-type structural proteins (C-E3-E2-6K-E1) of WEEV (strain CBA87), both alone and in combination with the VEEVW _T and EEEVW _T plasmids (WT combination). For this, groups of ten female BALB/c mice were vaccinated by PMED three times at 3- week intervals with 4 μg of the WEEVW _T plasmid or a WT combination comprising 4 μg each of the WEEVW _T? VEEVW _T , and EEEVW _T plasmids. A negative control group received 4 μg of the plasmid vector with no insert three times at 3 -week intervals by PMED. A positive control group was vaccinated three times at 3 -week intervals with 0.5 ml of the formalin-inactivated WEEV IND vaccine delivered by subcutaneous injection. Serum samples obtained 3 weeks after each vaccination were assayed for total anti-WEEV IgG antibodies by ELISA, and serum samples obtained three weeks after the third and final vaccination were also assayed for WEEV-neutralizing antibodies by PRNT. The mean ELISA titers of mice vaccinated with the WEEVW _T plasmid alone were higher than those of mice vaccinated with the WT combination after each of the three vaccinations (Fig. 23A). Mice vaccinated with the WEEVW _T plasmid alone or with the WT combination displayed similar low PRNT titers. To assess vaccine efficacy, the mice from all groups were challenged four weeks after the final vaccination with about 10 ⁴ PFU of WEEV strain CBA87 administered by the aerosol route. The survival rate of the mice vaccinated with WEEVW _T alone was 80%, while 50% of those receiving the WT composition survived the challenge (Fig. 23B). Mice vaccinated with the WEEV IND were protected at a level of 90%.

[131] To determine whether the WEEV DNA vaccine could be improved by

employing the same strategy that proved successful for the VEEV and EEEV DNA vaccines, the wild type 26S structural genes minus the capsid sequence (E3-E2-6K- El) of WEEV strain CBA87 (Genbank accession number DQ432026) were codon optimized by subjecting these genes to a GeneOptimizer™ bioinformatic algorithm (Geneart, Regensburg, Germany) for mammalian expression, and the codon optimized genes were synthesized using methods known in the art. The WEEVco plasmid was constructed by cloning these synthesized codon optimized genes into the Notl and BglW restriction sites of pWRG7077 using methods known in the art. [132] To determine if the WEEVco vaccine construct showed improved expression of the envelope glycoproteins as compared to the previous WEEV _WT construct, the proteins expressed in transiently transfected mammalian cells were detected by immune precipitation assay. The expression of the E2 and El envelope glycoproteins from the EEEVco DNA was increased approximately tenfold relative to that of the VEEVW _T construct in this assay (Fig. 24).

[133] In an initial study to determine whether the immunogenicity of the WEEVco plasmid was improved relative to the WEEVW _T plasmid, groups of six female BALB/c mice were vaccinated by i.m. EP three times at 3-week intervals with 25 μg, 5 μg, or 1 μg of DNA. A negative control group received 25 μg of the plasmid vector with no insert. Serum samples obtained 3 weeks after each vaccination were assayed for total anti-WEEV IgG antibodies by ELISA and for WEEV-neutralizing antibodies by PRNT. The mean ELISA titers of mice vaccinated with the WEEVco plasmid were significantly higher than those of mice vaccinated with the WEEVW _T construct for the 5 μg and 1 μg dose groups after each of the three vaccinations (Fig. 25 A). The mean PRNT go titer elicited by mice vaccinated with the WEEVco plasmid was significantly higher than that observed for mice vaccinated with the WEEVW _T construct for all dose groups at all days (Fig. 25B). In addition, the mean ELISA and PRNT titers of mice vaccinated with the WEEVco plasmid were not significantly different after two and three vaccinations for all dose groups.

[134] To determine the protective efficacy of the WEEVco DNA vaccine both alone and in the multivalent EEV combination (combination composition), groups of ten female BALB/c mice were vaccinated by i.m. EP twice at a 3-week interval with 5 μg of the WEEVco plasmid or a combination composition comprising 5 μg each of the WEEVco _? VEEVco, and EEEVco plasmids. A negative control group received the plasmid vector with no insert, and a positive control group received the formalin- inactivated WEEV IND vaccine. Serum samples obtained 3 weeks after each vaccination were assayed for total anti-WEEV IgG antibodies by ELISA and for WEEV-neutralizing antibodies by PRNT. The mean ELISA titers of mice vaccinated with the WEEVco plasmid alone were not significantly different from those of mice vaccinated with the combination composition after each of the vaccinations (Fig. 26A). In addition, the mean ELISA titers of mice receiving the individual WEEVco plasmid or the combination composition were higher than that of the WEEV IND after each of the vaccinations. The mean PRNT go titers elicited with the WEEVco plasmid alone were significantly higher than those for the combination composition or the WEEV IND vaccine after each vaccination, and those of the combination composition were lower than those of the WEEV IND after each of the vaccinations (Fig. 26B). To assess vaccine efficacy, the mice from all groups were challenged four weeks after the second and final vaccination with about 10 ⁴ PFU of WEEV strain CBA87 administered by the aerosol route. Surprisingly, mice vaccinated with the individual WEEVco plasmid and with the combination composition were completely protected from challenge (Fig. 26). In contrast, only 30% of mice receiving two vaccinations with the WEEV IND vaccine survived the challenge.

[135] WEEV DNA VACCINE IN RABBITS. TO assess the immunogenicity of the

WEEVco DNA vaccine alone and in the multivalent EEV combination (combination composition) in another species, female New Zealand White rabbits (N= 5) were vaccinated with 500 μg of the WEEVco plasmid or a combination composition comprising 500 μg each of the WEEVco _? VEEVco, and EEEVco plasmids delivered by i.m. EP on days 0, 28, and 230, and their antibody responses to WEEV were measured on days 0, 28, 42, 230, 266, and 349 by PRNT. The rabbits of both groups produced high titers of WEEV-neutralizing antibodies after a single vaccination and upon receiving a second vaccination, their mean PRNTgo titers were significantly boosted (Fig. 28). The PRNTgo titer of rabbits receiving the combination composition was lower than those of rabbits receiving the individual WEEVco plasmid on all days, yet they still remained remarkably high. In addition, the mean PRNTgo titers of both groups were boosted after an additional DNA vaccination performed on day 230.

[136] WEEV DNA VACCINE IN CYNOMOLGUS MACAQUES . The immunogenicity and protective efficacy of the WEEVco plasmid, both alone and in the multivalent EEV combination (combination composition), was assessed using an established model of WEEV infection of nonhuman primates. See Reed et al. (2005) J Infect Dis

192: 1 173-82. Groups of four adult male cynomolgus macaques were vaccinated three times at 4-week intervals with each animal receiving 500 μg of the empty vector DNA plasmid, 500 μg of the WEEVco plasmid, or a combination composition comprising 500 μg each of the WEEVco, VEEVco, and EEEVco plasmids delivered by i.m. EP. Serum samples collected 4 weeks after each vaccination were analyzed for WEEV- neutralizing antibodies by PRNT. Similar mean neutralizing antibody titers were elicited in macaques after a single vaccination with the individual WEEVco plasmid and with the combination composition, and the titers of both groups were significantly and similarly boosted after the second vaccination (Fig. 29). The day 85 mean titers of these groups are currently being determined. The vaccinated macaques will be challenged with about 1.0 x 10 ⁸ PFU of WEEV CBA87 at eight weeks after the third and final vaccination, and the protective efficacy will be evaluated in a similar manner to that previously done for VEEV and WEEV.

[137] Taken together, these results indicate that codon-optimized sequences that encode structural proteins of EEVs and exclude sequences that encode the capsid proteins of EEVs elicit robust and durable protective immune responses with low DNA doses and few vaccinations. These results also indicate that EP delivery is an effective method to elicit immunity in subjects, with a capacity large enough to accommodate more than one EEV nucleic acid molecule in a single administration. Therefore, the present invention provides compositions which comprise at least one codon-optimized sequence that encodes one or more structural proteins except the capsid protein of a given EEV, such as EEEV, VEEV or WEEV. In some

embodiments, the compositions comprise more than one codon-optimized sequence that encodes one or more structural proteins except the capsid protein of a given EEV. In these embodiments, the EEV may be the same or different. In some embodiments, the present invention provides combination compositions which comprise, consists essentially of, or consists of VEEVco, EEEVco, and WEEVco nucleic acid molecules and treatment methods using such.

[138] As used herein, the term "comprising" is used in its conventional sense to indicate that the given composition (or method) may include other ingredients (or steps). As used herein, the term "consisting of is used in its conventional sense to indicate that the given composition (or method) may not include any additional ingredients (or steps). As used herein, the phrase "consisting essentially of indicates that the given composition (or method) may include other ingredients (or steps) so long as the additional ingredients (or steps) do not materially change the biological and/or chemical activity (or results) of the specified ingredients (or steps). For example, a composition which consists essentially of VEEVco, EEEVco, and

WEEVco ^ma y contain additional ingredients so long as the additional ingredients do not change the biological activity, e.g. immunogenicity, of VEEVco, EEEVco, and WEEVco as compared to a composition which consists of VEEVco, EEEVco, and WEEVco- Similarly, a method which consists essentially of step A, step B, and step C, may include additional steps so long as the additional steps do not materially change the results of performing only steps A, B, and C.

[139] As used herein, an "EEVco polynucleotide" refers to a nucleotide sequence which encodes a plurality of structural proteins, except the capsid, of an equine encephalitis virus selected from the group consisting of VEEV, EEEV and WEEV. In some embodiments, the nucleotide sequence is codon-optimized for mammalian, preferably human, expression. In some preferred embodiments, the nucleotide sequence has at least about 85%, preferably about 90%, more preferably about 95%, or most preferably about 99% or more sequence identity to SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9. In some preferred embodiments, the nucleotide sequence is selected from the group consisting of SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9. As used herein, the terms "polynucleotide", "nucleic acid molecule", and "nucleotide sequence" are used interchangeably.

[140] As set forth herein, where a first set of variables are set forth and then a

second set of variables are indicated for the first set of variables, such a description is to be construed as if the second set of variables was explicitly indicated for each member of the first set of variables. For example, in the above paragraph, the third sentence shall be construed as explicitly indicating "In some preferred embodiments, the nucleotide sequence has at least about 85%, preferably about 90%, more preferably about 95%, or most preferably about 99% or more sequence identity to SEQ ID NO: l, or in some preferred embodiments, the nucleotide sequence has at least about 85%, preferably about 90%, more preferably about 95%, or most preferably about 99% or more sequence identity to SEQ ID NO:3, or in some preferred embodiments, the nucleotide sequence has at least about 85%, preferably about 90%), more preferably about 95%, or most preferably about 99% or more sequence identity to SEQ ID NO:5, or in some preferred embodiments, the nucleotide sequence has at least about 85%, preferably about 90%, more preferably about 95%, or most preferably about 99% or more sequence identity to SEQ ID NO:7, or in some preferred embodiments, the nucleotide sequence has at least about 85%, preferably about 90%), more preferably about 95%, or most preferably about 99% or more sequence identity to SEQ ID NO:8, or in some preferred embodiments, the nucleotide sequence has at least about 85%, preferably about 90%, more preferably about 95%, or most preferably about 99% or more sequence identity to SEQ ID NO:9". In other words, all possible permutations are to be considered as being explicitly set forth. It is noted that this abbreviated manner of setting forth the embodiments of the instant invention is being used in the interests of reducing extra page fees.

[141] As used herein "EEVco plasmid" refers to plasmid comprising, consisting essentially of, or consisting of a vector sequence and an EEVco polynucleotide as described herein. In other words, an EEVco plasmid refers to an EEVco

polynucleotide that is contained within a vector, such as an expression vector known in the art. In some embodiments, the EEVco plasmids of the present invention that comprise an EEVco sequence inserted therein. Examples of EEVco plasmids include VEEVco, EEEVco, and WEEVco plasmids as set forth in Figs. 30A-30C.

[142] As disclosed herein, the present invention provides VEEVco, EEEVco, and

WEEVco plasmids which may be used as DNA vaccines. The EEVco plasmids of the present invention express the structural genes of the 26S subgenomic mRNA of these viruses minus the capsid protein coding region (E3-E2-6K-E1) that have been optimized to include several features known to increase mammalian expression. The VEEVco, EEEVco, and WEEVco plasmids express the structural genes of VEEV subtype IAB strain Trinidad Donkey, EEEV strain FL91-4679, and WEEV CBA87, respectively, that were adapted to the codon bias of Homo sapiens genes. The codon adaptation index (CAI), a measure of how well the codons of the optimized sequence match the codon usage preference of the target organism, was raised from 0.74 to 0.98 for the VEEV genes, from 0.71 to 0.98 for the EEEV genes, and from 0.70 to 0.98 for the WEEV genes. In addition, regions of very high (>80%) or very low (<30%) guanine-cytosine (GC) content were avoided in the genes where possible. The average GC content was increased from 51% to 65% for the VEEV genes and from 50%) to 65%o for the EEEV and WEEV genes. The optimization process also included the avoidance of negatively cis-acting motifs in the genes which can hamper expression in mammals. For the VEEV genes, 9 prokaryotic inhibitory motifs, 1 polyadenylation site, 3 consensus splice donor sites, 4 cryptic splice donor sites, and 5 RNA instability motifs were avoided in the optimized gene sequence. For the EEEV genes, 6 prokaryotic inhibitory motifs, 1 polyadenylation site, 2 consensus splice donor sites, 3 cryptic splice donor sites, and 3 RNA instability motifs were avoided in the optimized gene sequence. For the WEEV genes, 9 prokaryotic inhibitory motifs, 1 polyadenylation site, 2 consensus splice donor sites, 4 cryptic splice donor sites, and 4 RNA instability motifs were avoided in the optimized gene sequence. In addition, Kozak sequence was introduced into the leader sequence of each of the optimized VEEV, EEEV, and WEEV structural gene open reading frames to increase translational initiation. Finally, two STOP codons were added at the ends of the optimized VEEV, EEEV, and WEEV structural gene open reading frames to ensure efficient termination. The optimized VEEV, EEEV, and WEEV structural gene open reading frames were then synthesized by Geneart, Inc. (Regensburg, Germany) and individually inserted between the Not/ and Bgl// restriction sites of plasmid backbone pWRG7077 to create the final VEEVco, EEEVco, and WEEVco plasmids that are exemplified herein. See Figs. 30A-30C and the respective sequences. The pWRG7077 plasmid backbone contains the human cytomegalovirus immediate early (CMV IE) promoter with its associated Intron A, a bovine growth hormone transcription terminator and polyadenylation signal (BGH pA), a pUC19 origin of replication (ori), and a kanamycin resistance marker (KanR).

[143] As used herein, an "EEVco antibody" refers to an antibody raised against an

EEVco plasmid and/or an EEVco polynucleotide.

[144] As used herein, a "corresponding EEVW _T control" refers to the wild type form of the item referenced. For example, a corresponding EEVW _T control of VEEVco is the corresponding wild type sequence, VEEVW _T - Similarly, the corresponding EEVW _T control of a WEEVco plasmid, is a plasmid which has the same vector sequence as the WEEVco plasmid except that the polynucleotide contained therein is WEEVW _T - As another example, a corresponding EEVW _T control of a combination composition (e.g. a composition comprising VEEVco, EEEVco, and WEEVco polynucleotides) is a composition comprising VEEV _WT , EEEV _WT , and WEEV _WT polynucleotides (WT combination) in the same amounts as that provided in the combination composition. It is important to note the differences between the WT, COCAP, and CO designations as used herein. The WT plasmids have inserts consisting of the wild-type full structural gene locus (C-E3-E2-6K-E1) of VEEV, EEEV or WEEV. The COCAP plasmids contain the full structural gene locus (C-E3- E2-6K-E1) of VEEV, EEEV, or WEEV that has been codon optimized. The CO plasmids contain the codon optimized structural gene locus of VEEV, EEEV, or WEEV minus the capsid (C) protein coding region (E3-E2-6K-E1). Therefore, the COCAP and CO plasmids are identical except for the lack of the codon optimized capsid coding region in the CO plasmids. [145] As used herein, a "subject" refers to a mammal, preferably a human who may be a patient, e.g. under the care of a physician.

[146] The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in a subject. A pharmaceutical composition generally comprises an effective amount of an active agent, e.g. an EEVco plasmid, an EEVco

polynucleotide, or an EEVco antibody according to the present invention, and a pharmaceutically acceptable carrier, e.g. a buffer, adjuvant, and the like. The term "effective amount" refers to a dosage or amount sufficient to produce a desired result. The desired result may comprise an objective or subjective improvement in the recipient of the dosage or amount, e.g. long-term survival, decrease in viremia, effective prevention of a disease state, and the like.

[147] The EEVco plasmid, EEVco polynucleotide, or EEVco antibody of the present invention may be administered, preferably in the form of pharmaceutical

compositions, to a subject. Preferably the subject is mammalian, more preferably, the subject is human. Preferred pharmaceutical compositions are those comprising at least one immunogenic composition against VEEV, EEEV, WEEV, or a combination thereof, in an immunogenic amount or a therapeutically effective amount, and a pharmaceutically acceptable vehicle. The immunogenic composition may be an active immunizing agent, such as an EEVco plasmid of the present invention, or a passive immunizing agent, such as an EEVco antibody. The immunogenic composition may elicit an immune response that need not be protective or the immunogenic composition may provide passive immunity. EEVco vaccines according to the present invention elicit a local or systemic immune response that is protective against subsequent challenge with an EEV virus, such as VEEV, EEEV, WEEV, or a combination thereof. Conventional methods in the art may be used to determine the feasibility of using the nucleic acid molecules of the present invention as vaccines against EEV infection. Accordingly, as used herein, an "immunogenic composition" can refer to vaccines as well as antibodies. A protective immune response may be complete or partial, i.e. a reduction in symptoms as compared with an unvaccinated mammal.

[148] Thus, the present invention provides immunogenic compositions comprising at least one EEVco plasmid and/or at least one EEVco polynucleotide according to the present invention and/or at least one EEVco antibody of the present invention that may be used to immunize a subject against one or more EEV by methods known in the art. As used herein, an "immunogenic amount" is an amount that is sufficient to elicit an immune response in a subject and depends on a variety of factors such as the immunogenicity of the EEVco plasmid, the manner of administration, the general state of health of the subject, and the like. The typical immunogenic amounts for initial and boosting immunization for therapeutic or prophylactic administration ranges from about 0.01 mg to about 200 mg, preferably about 0.1-5.0 mg, more preferably about 0.5-2.0 mg, per about 65-70 kg body weight of a subject. For example, the typical immunogenic amount for initial and boosting immunization for therapeutic or prophylactic administration for a human subject ranges from about 0.01 mg to about 200 mg, preferably about 0.1-5.0 mg, more preferably about 0.5-2.0 mg, per about 65-70 kg body weight of a subject. Examples of suitable immunization protocols include initial immunization injections at time 0 and 4 or initial

immunization injections at 0, 4, and 8 weeks, which initial immunization injections may be followed by further booster injections at 1 or 2 years.

[149] As used herein, a "therapeutically effective amount" refers to an amount that may be used to treat, prevent, or inhibit a given condition, such as an EEV infection, in a subject as compared to a control. Again, the skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including the severity of EEV exposure, previous treatments, the general health and age of the subject, and the like. A therapeutically effective amount may be readily determined by conventional methods known in the art. It should be noted that treatment of a subject with a therapeutically effective amount of a given substance, e.g. an EEVco plasmid, an EEVco polynucleotide, or an EEVco antibody according to the present invention, may be administered as a single dose or as a series of several doses.

[150] The pharmaceutical compositions may include an adjuvant. As used herein, an "adjuvant" refers to any substance which, when administered with or before a pharmaceutically active agent, such as an EEVco plasmid, aids the pharmaceutically active agent in its mechanism of action. Thus, an adjuvant in a vaccine is a substance that aids the immunogenic composition in eliciting an immune response. Suitable adjuvants include incomplete Freund's adjuvant, alum, aluminum phosphate, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N- acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, nor-MDP), N- acetylmuramyl-Lalanyl-D-isoglutaminyl-L-alanine-2-( 1 ' -2 ' -dipa-lmitoyl-sn-glycero- 3-hydroxyphosphoryloxy)-ethylamine (CGP 19835 A, MTP-PE), and RIBI, which comprise three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (NPL+TDM+CWS) in a 2%

squalene/Tween 80 emulsion. The effectiveness of an adjuvant may be determined by conventional methods in the art.

[151] The compositions of the present invention may be administered to a subject by any suitable route including oral, transdermal, intranasal, inhalation, intramuscular, and intravascular administration. It will be appreciated that the preferred route of administration and pharmaceutical formulation will vary with the condition and age of the subject, the nature of the condition to be treated, the therapeutic effect desired, and the particular polypeptide, polynucleotide, or antibody used.

[152] As used herein, a "pharmaceutically acceptable vehicle" or "pharmaceutically acceptable carrier" refers to and includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration.

Pharmaceutically acceptable vehicles include those known in the art. See e.g.

REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY. 20 ^th ed. (2000) Lippincott Williams & Wilkins. Baltimore, MD, which is herein incorporated by reference.

[153] The pharmaceutical compositions of the present invention may be provided in dosage unit forms. "Dosage unit form" as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of

compounding such an active compound for the treatment of individuals.

[154] The formulations of the compositions of the present invention may be

optimized for increased stability and efficacy using methods known in the art. See e.g. Carra et al. (2007) Vaccine 25:4149-4158, which is herein incorporated by reference.

[155] Toxicity and therapeutic efficacy of the polynucleotides and antibodies

according to the instant invention and compositions thereof can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. For example, one may determine the lethal dose, LC ₅₀ (the dose expressed as concentration x exposure time that is lethal to 50% of the population) or the LD ₅₀ (the dose lethal to 50% of the population), and the ED ₅₀ (the dose therapeutically effective in 50% of the population) by conventional methods in the art. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD ₅₀ /ED ₅₀ . Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[156] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC ₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[157] The present invention also provides EEVco plasmids, EEVco polynucleotides,

EEVco antibodies, and/or compositions thereof provided in kits along with

instructions for use. A kit comprising a pharmaceutical composition may include the pharmaceutical composition as a single dose or multiple doses. The kit may include a device for delivering one or more EEVco plasmids, one or more EEVco

polynucleotides, one or more EEVco antibodies, and/or compositions thereof. The device may be a multi-chambered syringe for intramuscular delivery, a microneedle or set of microneedle arrays for transdermal delivery, a small balloon for intranasal delivery, or a small aerosol generating device for delivery by inhalation.

[158] To the extent necessary to understand or complete the disclosure of the present invention, all publications, patents, and patent applications mentioned herein are expressly incorporated by reference therein to the same extent as though each were individually so incorporated.

Having thus described exemplary embodiments of the present invention, it should be noted by those skilled in the art that the within disclosures are exemplary only and that various other alternatives, adaptations, and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments as illustrated herein, but is only limited by the following claims.