VIOLIN: Vaccine Investigation and Online Information Network

VEE Virus

Table of Contents

General Information
Vaccine Related Pathogen Genes
1. POLS_EEVVT Structural polyprotein (p130)
2. TC-83 (Other)
3. 26S mRNA (Protective antigen)
4. C-E3-E2-E1-6K (Protective antigen)
5. E1 glycoprotein (Protective antigen, Virmugen)
6. E2 envelope protein (Protective antigen)
7. V antigen from Y. pestis (Protective antigen)
8. PE2 (Virmugen)
Vaccine Information
References

I. General Information

1. NCBI Taxonomy ID:

11036

2. Disease:

Venezuelan equine encephalitis

3. Introduction

Venezuelan equine encephalitis virus (VEEV) is a naturally emerging disease threat and a highly developed biological weapon. VEEV is the most important human and equine pathogen of the New World alphaviruses (Togaviridae: Alphavirus). VEEV causes periodic outbreaks of febrile and neurological disease. VEE was first recognized as a disease of horses, mules, and donkeys in northern South America during the 1930s. VEEV, was first isolated in 1938 from the brains of fatal equine cases in Yaracuy State, Venezuela. VEEV is a spherical virus 70 nm in diameter and a messenger-sense, single-stranded RNA genome approximately 11,400 nucleotides in length (Weaver et al., 2004).

4. Microbial Pathogenesis

In equines and humans, VEEV causes inapparent to acute encephalitis. Enzootic VEE strains in subtypes I-E, II, III, and IV are avirulent for equines and generally produce only low titered viremia and little or no illness. However, at least some of the enzootic viruses can be pathogenic for humans and have caused fatal disease. The incubation period in human VEEV infection is usually 2–5 days. VEE occurs in all human age groups without sex bias. Children are more likely to develop fatal encephalitis than adults. VEEV also causes birth defects, abortions and stillbirths in pregnant women. In experimentally infected equines and rodents, VEEV causes severe myeloid depletion in bone marrow and lymphocyte destruction in lymph, nodes and spleen. Encephalitis is accompanied by a wide range of histopathology, from mild neutrophilic infiltration to neuronal degeneration, necrotizing vasculitis, and Purkinje cell destruction. In mice, VEEV appears to reach the brain via the olfactory nerve, seeded by viremia (Weaver et al., 2004).

VEEV encodes four nonstructural proteins (nsP1–4) and three structural proteins: the capsid and the E1 and E2 envelope glycoproteins. The E2 protein forms spikes on the surface of the virion. The E1 protein lies adjacent to the host cell–derived lipid envelope. VEEV can use the laminin binding protein as a receptor for entry into cells via receptor-mediated endocytosis. After fusion of virions with the membrane of endosomes at low pH via a hydrophobic amino acid sequence in the E1 protein, the genome is translated in the cytoplasm to generate the nonstructural polyprotein. Viral genome replication occurs on the cytoplasmic surface of endosomes. One molecule of genomic RNA interacts in the cytoplasm with 240 copies of the capsid protein to form a nucleocapsid. The envelope glycoproteins are inserted into the endoplasmic reticulum membrane and interact with nucleocapsids at the plasma membrane to initiate budding of virus particles on the surface of cells (Weaver et al., 2004).

Biological transmission of arthropod-borne VEEV involves initial infection of the mosquito midgut following ingestion of a viremic blood meal. Posterior midgut epithelial cells become infected first, followed by dissemination into the hemocoel and infection of secondary organs and tissues including the salivary glands. Virus maturation (budding) in midgut epithelial cells occurs exclusively on the basal margins adjacent to the basal lamina (Weaver et al., 2004).

The epizootic transmission cycle of VEEV actively involves equines as highly efficient amplification hosts. Although the vertebrate host range of epizootic VEEV strains is wide and includes humans, sheep, dogs, bats, rodents, and some birds, major epidemics in the absence of equine cases have never occurred despite the repeated occurrence of epizootics near major cities such as Maracaibo (Weaver et al., 2004).

Sylvatic rodents in the genera Sigmodon, Oryzomys, Zygodontomys, Heteromys, Peromyscus, and Proechimys are believed to be the principal reservoir hosts of most enzootic VEE complex viruses. They are frequently infected in nature. They also have high rates of immunity and develop moderate to high titered viremia (Weaver et al., 2004).

5. Host Ranges and Animal Models

VEE causes encephalitis in a wide range of vertebrate animals including humans, horses, mules, donkeys, sheep, dogs, bats, rodents, and some birds. Rodents have been frequently used in the laboratory for VEEV pathogenesis and vaccine studies. VEEV is also an arthropod-borne virus and can infect and replicate in mosquito (Weaver et al., 2004).

6. Host Protective Immunity

Cell-mediated immunity plays a predominant role in protection against VEEV. Immunity resulting from inactivated VEEV vaccines is short-lived and frequent boosters are required to maintain protection. Inactivated VEEV vaccines generate protective neutralizing immunity only after multiple inoculations (Weaver et al., 2004).

II. Vaccine Related Pathogen Genes

1. 26S mRNA

Gene Name : 26S mRNA
Sequence Strain (Species/Organism) : Venezuelan equine encephalitis virus
NCBI Gene ID : 2652924
NCBI Protein GI : 9626528
Locus Tag : VEEVgp3
Genbank Accession : AY973944
Protein Accession : NP_040824
Taxonomy ID : 11036
Gene Starting Position : 7531
Gene Ending Position : 11443
Gene Strand (Orientation) : +
Protein Name : mRNA
Protein pI : 8.88
Protein Weight : 129183.21
Protein Length : 1255

DNA Sequence : Show Sequence

>NC_001449.1:7531-11443 Venezuelan equine encephalitis virus, complete genome
AATGGACTACGACATAGTCTAGTCCGCCAAGATGTTCCCGTTCCAACCAATGTATCCGATGCAGCCAATG
CCCTATCGTAACCCGTTCGCGGCCCCGCGCAGGCCCTGGTTCCCCAGAACCGACCCTTTTCTGGCGATGC
AGGTGCAGGAATTAACCCGCTCGATGGCTAACCTGACGTTCAAGCAACGCCGGGACGCGCCACCTGAGGG
GCCACCTGCTAAGAAACCTAAGAGGGAGGCCCCGCAAAAGCAAAAAGGGGGAGGCCAAGGGAAGAAGAAG
AAGAACCAGGGGAAGAAGAAGGCCAAGACGGGGCCGCCTAATCCGAAGGCACAGAGTGGAAACAAGAAGA
AGCCCAACAAGAAACCAGGCAAGAGACAGCGCATGGTCATGAAATTGGAATCTGACAAGACATTCCCAAT
TATGCTGGAAGGGAAGATTAACGGCTACGCTTGCGTGGTCGGAGGGAAGTTATTCAGGCCGATGCACGTG
GAAGGCAAGATCGACAACGACGTTCTGGCCGCACTTAAGACGAAGAAAGCATCCAAATATGATCTTGAGT
ATGCAGATGTGCCACAGAACATGCGGGCCGATACATTCAAGTACACCCATGAGAAGCCCCAAGGCTATTA
CAGCTGGCATCATGGAGCAGTCCAATATGAAAATGGGCGTTTCACGGTGCCAAAAGGAGTTGGGGCCAAG
GGAGACAGCGGAAGACCCATTCTGGATAATCAGGGACGGGTGGTCGCTATTGTGCTGGGAGGTGTGAATG
AAGGATCTAGGACAGCCCTTTCAGTCGTCATGTGGAACGAGAAGGGAGTAACTGTGAAGTATACTCCGGA
GAACTGCGAGCAATGGTCACTAGTGACCACTATGTGCCTGCTCGCCAATGTGACGTTCCCATGTGCCGAA
CCACCAATTTGCTACGACAGAAAACCAGCAGAGACTTTGGCCATGCTCAGCGTTAACGTTGACAACCCGG
GCTACGATGAGCTGCTGGAAGCAGCTGTTAAGTGCCCCGGAAGAAAAAGGAGATCTACCGAGGAGCTGTT
TAAGGAGTATAAGCTAACGCGCCCTTACATGGCCAGATGCATCAGATGTGCCGTTGGGAGCTGCCATAGT
CCAATAGCAATTGAGGCAGTGAAGAGCGACGGGCACGACGGCTATGTTAGACTTCAGACTTCCTCGCAGT
ATGGCCTGGATTCCTCTGGCAACTTAAAGGGAAGGACTATGCGGTATGATATGCACGGGACCATTGAAGA
GATACCACTACATCAAGTGTCACTCCACACATCTCGCCCGTGTCACATTGTGGATGGGCATGGTTATTTT
CTGCTTGCTAGGTGCCCGGCAGGGGACTCCATCACCATGGAATTTAAGAAAGGTTCAGTCACACACTCCT
GCTCAGTGCCGTATGAAGTGAAATTTAATCCTGTAGGCAGAGAACTCTACACTCATCCACCAGAACACGG
AGCAGAGCAAGCGTGCCAAGTCTACGCGCACGATGCACAGAACAGAGGAGCTTATGTCGAGATGCACCTC
CCGGGCTCAGAAGTGGACAGCAGTTTGATTTCCTTGAGCGGCAGTTCAGTCACCGTGACACCTCCTGTCG
GGACTAGCGCCTTGGTGAAATGCAAGTGCGGCGGCACAAAGATCTCCGAAACCATCAACAAGGCAAAACA
GTTCAGCCAGTGCACAAAGAAGGAGCAGTGCAGAGCATATCGACTGCAGAATGACAAGTGGGTGTATAAT
TCTGACAAACTGCCCAAAGCAGCGGGAGCCACCCTAAAAGGAAAACTACACGTCCCGTTCTTGCTGGCAG
ACGGCAAATGCACCGTGCCTCTAGCACCGGAACCTATGATAACCTTCGGTTTCCGATCAGTGTCACTGAA
ACTGCACCCTAAGAATCCCACATATCTGACCACTCGCCAACTTGCTGATGAGCCTCATTACACGCACGAG
CTCATATCTGAACCAGCTGTTAGGAATTTTACCGTCACTGAAAAGGGGTGGGAGTTTGTATGGGGAAACC
ATCCGCCGAAAAGGTTTTGGGCACAGGAAACAGCACCCGGAAATCCACATGGGCTGCCACATGAGGTGAT
AACTCATTATTACCACAGATACCCTATGTCCACCATCCTGGGTTTGTCAATTTGCGCCGCCATTGTAACC
GTTTCCGTTGCAGCGTCCACCTGGCTGTTTTGCAAATCCAGAGTTTCGTGCCTAACTCCTTACCGGCTAA
CACCTAACGCCAGGATGCCGCTTTGCCTGGCCGTGCTTTGCTGCGCCCGCACTGCCCGGGCCGAGACCAC
CTGGGAGTCCTTGGATCACCTATGGAACAATAACCAACAGATGTTCTGGATTCAATTGCTGATCCCTCTG
GCCGCCTTGATTGTAGTGACTCGCCTGCTCAAGTGCGTGTGCTGTGTAGTGCCTTTTTTAGTCGTGGCCG
GCGCCGCAGGCGCCGGCGCCTACGAGCACGCGACCACGATGCCGAGCCAAGCGGGAATCTCGTATAACAC
CATAGTCAACAGAGCAGGCTACGCGCCACTCCCTATCAGCATAACACCAACAAAGATCAAGCTGATACCC
ACAGTGAACTTGGAGTACGTCACCTGCCACTACAAAACAGGAATGGATTCACCAGCCATCAAATGCTGCG
GATCTCAGGAATGTACTCCAACTAACAGGCCTGATGAACAGTGCAAAGTCTTCACAGGGGTTTACCCGTT
CATGTGGGGAGGTGCATATTGCTTTTGCGACACTGAGAATACTCAGGTCAGCAAGGCCTACGTAATGAAA
TCTGACGACTGCCTTGCGGATCATGCTGAAGCATACAAAGCGCACACAGCCTCAGTGCAGGCGTTCCTCA
ACATCACAGTGGGGGAACACTCTATTGTGACCACCGTGTATGTGAATGGAGAAACTCCTGTGAACTTCAA
TGGGGTCAAACTAACTGCAGGTCCACTTTCCACAGCTTGGACACCCTTTGACAGAAAAATCGTGCAGTAT
GCCGGGGAGATCTATAATTACGATTTTCCTGAGTATGGGGCAGGACAACCAGGAGCATTTGGAGACATAC
AATCCAGAACAGTCTCAAGCTCAGATCTGTATGCCAATACCAACCTAGTGCTGCAGAGACCCAAAGCAGG
AGCGATCCATGTGCCATACACTCAGGCACCATCGGGTTTTGAGCAATGGAAGAAAGATAAAGCTCCGTCA
TTGAAATTCACCGCCCCTTTCGGATGCGAAATATATACAAACCCCATTCGCGCCGAAAATTGTGCTGTAG
GGTCAATTCCATTAGCCTTTGACATTCCCGACGCCTTGTTCACCAGGGTGTCAGAAACACCGACACTTTC
AGCGGCCGAATGCACTCTTAACGAGTGCGTGTATTCATCCGACTTTGGCGGGATCGCCACGGTCAAGTAT
TCGGCCAGCAAGTCAGGCAAGTGCGCAGTCCATGTGCCATCAGGGACTGCTACCCTAAAAGAAGCAGCAG
TCGAGCTAACCGAGCAAGGGTCGGCGACCATTCATTTCTCGACCGCAAATATCCACCCGGAGTTCAGGCT
CCAAATATGCACATCATATGTCACGTGCAAAGGTGATTGTCACCCCCCGAAAGACCACATTGTGACACAC
CCCCAGTATCACGCCCAAACATTTACAGCCGCGGTGTCAAAAACCGCGTGGACGTGGTTAACATCCCTGC
TGGGAGGATCGGCCGTAATTATTATAATTGGCTTAGTGCTGGCTACTATTGTGGCCATGTACGTGCTGAC
CAACCAGAAACATAATTGAACATAGCAGCAATTGGCAAGCTGCTTATATAGAACTTGCGGCGATTGGCAT
GCCGCTTTAAAATTTTATTTTATTTTCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTT

Protein Sequence : Show Sequence

>NP_040824.1 structural polyprotein precursor [Venezuelan equine encephalitis virus]
MFPFQPMYPMQPMPYRNPFAAPRRPWFPRTDPFLAMQVQELTRSMANLTFKQRRDAPPEGPPAKKPKREA
PQKQKGGGQGKKKKNQGKKKAKTGPPNPKAQSGNKKKPNKKPGKRQRMVMKLESDKTFPIMLEGKINGYA
CVVGGKLFRPMHVEGKIDNDVLAALKTKKASKYDLEYADVPQNMRADTFKYTHEKPQGYYSWHHGAVQYE
NGRFTVPKGVGAKGDSGRPILDNQGRVVAIVLGGVNEGSRTALSVVMWNEKGVTVKYTPENCEQWSLVTT
MCLLANVTFPCAEPPICYDRKPAETLAMLSVNVDNPGYDELLEAAVKCPGRKRRSTEELFKEYKLTRPYM
ARCIRCAVGSCHSPIAIEAVKSDGHDGYVRLQTSSQYGLDSSGNLKGRTMRYDMHGTIEEIPLHQVSLHT
SRPCHIVDGHGYFLLARCPAGDSITMEFKKGSVTHSCSVPYEVKFNPVGRELYTHPPEHGAEQACQVYAH
DAQNRGAYVEMHLPGSEVDSSLISLSGSSVTVTPPVGTSALVKCKCGGTKISETINKAKQFSQCTKKEQC
RAYRLQNDKWVYNSDKLPKAAGATLKGKLHVPFLLADGKCTVPLAPEPMITFGFRSVSLKLHPKNPTYLT
TRQLADEPHYTHELISEPAVRNFTVTEKGWEFVWGNHPPKRFWAQETAPGNPHGLPHEVITHYYHRYPMS
TILGLSICAAIVTVSVAASTWLFCKSRVSCLTPYRLTPNARMPLCLAVLCCARTARAETTWESLDHLWNN
NQQMFWIQLLIPLAALIVVTRLLKCVCCVVPFLVVAGAAGAGAYEHATTMPSQAGISYNTIVNRAGYAPL
PISITPTKIKLIPTVNLEYVTCHYKTGMDSPAIKCCGSQECTPTNRPDEQCKVFTGVYPFMWGGAYCFCD
TENTQVSKAYVMKSDDCLADHAEAYKAHTASVQAFLNITVGEHSIVTTVYVNGETPVNFNGVKLTAGPLS
TAWTPFDRKIVQYAGEIYNYDFPEYGAGQPGAFGDIQSRTVSSSDLYANTNLVLQRPKAGAIHVPYTQAP
SGFEQWKKDKAPSLKFTAPFGCEIYTNPIRAENCAVGSIPLAFDIPDALFTRVSETPTLSAAECTLNECV
YSSDFGGIATVKYSASKSGKCAVHVPSGTATLKEAAVELTEQGSATIHFSTANIHPEFRLQICTSYVTCK
GDCHPPKDHIVTHPQYHAQTFTAAVSKTAWTWLTSLLGGSAVIIIIGLVLATIVAMYVLTNQKHN

Molecule Role : Protective antigen
Molecule Role Annotation : Representative variants from a library in which the E1 gene from VEEV IA/B was held constant and only the E2 genes of the five parent viruses were recombined elicited significantly increased neutralizing antibody titers to VEEV IA/B compared to the parent DNA vaccine and provided improved protection against aerosol VEEV IA/B challenge in BALB/c mice (Dupuy et al., 2009).
Related Vaccine(s): VEE virus DNA vaccine encoding 26S

2. C-E3-E2-E1-6K

Gene Name : C-E3-E2-E1-6K
Sequence Strain (Species/Organism) : Venezuelan equine encephalitis virus (strain TC-83)
NCBI Protein GI : 130559
Other Database IDs : CDD:279312
CDD:279849
CDD:279311
CDD:279870
Taxonomy ID : 11037
Gene Strand (Orientation) : ?
Protein Name : Structural polyprotein
Protein pI : 8.56
Protein Weight : 137728.43
Protein Length : 1776
Protein Note : p130

Protein Sequence : Show Sequence

>sp|P05674.1|POLS_EEVV8 RecName: Full=Structural polyprotein; AltName: Full=p130; Contains: RecName: Full=Capsid protein; AltName: Full=Coat protein; Short=C; Contains: RecName: Full=Precursor of protein E3/E2; AltName: Full=p62; AltName: Full=pE2; Contains: RecName: Full=Assembly protein E3; Contains: RecName: Full=Spike glycoprotein E2; AltName: Full=E2 envelope glycoprotein; Contains: RecName: Full=6K protein; Contains: RecName: Full=Spike glycoprotein E1; AltName: Full=E1 envelope glycoprotein
MFPFQPMYPMQPMPYRNPFAAPRRPWFPRTDPFLAMQVQELTRSMANLTFKQRRDAPPEGPSAKKPKKEA
SQKQKGGGQGKKKKNQGKKKAKTGPPNPKAQNGNKKKTNKKPGKRQRMVMKLESDKTFPIMLEGKINGYA
CVVGGKLFRPMHVEGKIDNDVLAALKTKKASKYDLEYADVPQNMRADTFKYTHEKPQGYYSWHHGAVQYE
NGRFTVPKGVGAKGDSGRPILDNQGRVVAIVLGGVNEGSRTALSVVMWNEKGVTVKYTPENCEQWSLVTT
MCLLANVTFPCAQPPICYDRKPAETLAMLSVNVDNPGYDELLEAAVKCPGRKRRSTEELFNEYKLTRPYM
ARCIRCAVGSCHSPIAIEAVKSDGHDGYVRLQTSSQYGLDSSGNLKGRTMRYDMHGTIKEIPLHQVSLYT
SRPCHIVDGHGYFLLARCPAGDSITMEFKKDSVRHSCSVPYEVKFNPVGRELYTHPPEHGVEQACQVYAH
DAQNRGAYVEMHLPGSEVDSSLVSLSGSSVTVTPPDGTSALVECECGGTKISETINKTKQFSQCTKKEQC
RAYRLQNDKWVYNSDKLPKAAGATLKGKLHVPFLLADGKCTVPLAPEPMITFGFRSVSLKLHPKNPTYLI
TRQLADEPHYTHELISEPAVRNFTVTEKGWEFVWGNHPPKRFWAQETAPGNPHGLPHEVITHYYHRYPMS
TILGLSICAAIATVSVAASTWLFCRSRVACLTPYRLTPNARIPFCLAVLCCARTARAETTWESLDHLWNN
NQQMFWIQLLIPLAALIVVTRLLRCVCCVVPFLVMAGAAAPAYEHATTMPSQAGISYNTIVNRAGYAPLP
ISITPTKIKLIPTVNLEYVTCHYKTGMDSPAIKCCGSQECTPTYRPDEQCKVFTGVYPFMWGGAYCFCDT
ENTQVSKAYVMKSDDCLADHAEAYKAHTASVQAFLNITVGEHSIVTTVYVNGETPVNFNGVKITAGPLST
AWTPFDRKIVQYAGEIYNYDFPEYGAGQPGAFGDIQSRTVSSSDLYANTNLVLQRPKAGAIHVPYTQAPS
GFEQWKKDKAPSLKFTAPFGCEIYTNPIRAENCAVGSIPLAFDIPDALFTRVSETPTLSAAECTLNECVY
SSDFGGIATVKYSASKSGKCAVHVPSGTATLKEAAVELTEQGSATIHFSTANIHPEFRLQICTSYVTCKG
DCHPPKDHIVTHPQYHAQTFTAAVSKTAWTWLTSLLGGSAVIIIIGLVLATIVAMYVLTNQKHN

Molecule Role : Protective antigen
Related Vaccine(s): VEE virus DNA vaccine pSTU-TRDF encoding VEEV E3–E2–6K

3. E1 glycoprotein

Gene Name : E1 glycoprotein
Sequence Strain (Species/Organism) : Venezuelan equine encephalitis virus
VO ID : VO_0011267
NCBI Protein GI : 798795
Other Database IDs : CDD:201874
Taxonomy ID : 11036
Protein Name : E1 glycoprotein
Protein pI : 7.54
Protein Weight : 3856.25
Protein Length : 114
Protein Note : Alphavirus E1 glycoprotein; pfam01589

Protein Sequence : Show Sequence

>AAA65907.1 E1 glycoprotein, partial [Venezuelan equine encephalitis virus]
WTWLTSLLGGSAVIIIIGLVLATIVAMYVLTNQKHN

Molecule Role : Protective antigen
Molecule Role Annotation : The monoclonal antibody against E1 glycoprotein protect outbred albino mice from lethal infection caused by the virulent strain Trd of the VEE virus (Agapov et al., 1994).
Additional Molecule Role : Virmugen
Additional Molecule Role Annotation : A VEE mutant with a PE2 cleavage signal mutation and a suppressor mutation in E1 is highly attenuated in mice and provides complete protection against challenge with wild type VEE (Davis et al., 1995).
Related Vaccine(s): VEE virus DNA vaccine VEEV IA/B parent encoding structural genes , VEE Virus PE2/E1 mutant vaccine

4. E2 envelope protein

Gene Name : E2 envelope protein
Sequence Strain (Species/Organism) : Venezuelan equine encephalitis virus
VO ID : VO_0011265
NCBI Protein GI : 25140296
Other Database IDs : CDD:279311
Taxonomy ID : 11036
Protein Name : E2 envelope protein
Protein pI : 8.59
Protein Weight : 44311.99
Protein Length : 503
Protein Note : putative

Protein Sequence : Show Sequence

>NP_741966.1 E2 envelope protein [Venezuelan equine encephalitis virus]
STEELFKEYKLTRPYMARCIRCAVGSCHSPIAIEAVKSDGHDGYVRLQTSSQYGLDSSGNLKGRTMRYDM
HGTIEEIPLHQVSLHTSRPCHIVDGHGYFLLARCPAGDSITMEFKKGSVTHSCSVPYEVKFNPVGRELYT
HPPEHGAEQACQVYAHDAQNRGAYVEMHLPGSEVDSSLISLSGSSVTVTPPVGTSALVKCKCGGTKISET
INKAKQFSQCTKKEQCRAYRLQNDKWVYNSDKLPKAAGATLKGKLHVPFLLADGKCTVPLAPEPMITFGF
RSVSLKLHPKNPTYLTTRQLADEPHYTHELISEPAVRNFTVTEKGWEFVWGNHPPKRFWAQETAPGNPHG
LPHEVITHYYHRYPMSTILGLSICAAIVTVSVAASTWLFCKSRVSCLTPYRLTPNARMPLCLAVLCCART
ARA

Molecule Role : Protective antigen
Molecule Role Annotation : Since the E2 amino-terminal sequence for all VEE subtype viruses is conserved, we tested the protective capacity in mice of passively transferred mAb 1A2B-10 and found it to protect from both epizootic and enzootic VEE virus challenge. Since horses are an important natural host for VEE virus, pep1-19 was used to immunize horses and was found to be immunogenic and to elicit virus-reactive antibody (Hunt and Roehrig, 1995).
Related Vaccine(s): VEE virus DNA vaccine VEEV IA/B parent encoding structural genes

5. PE2

Gene Name : PE2
Sequence Strain (Species/Organism) : Venezuelan equine encephalitis virus
NCBI Protein GI : 229558446
Other Database IDs : CDD:190039
CDD:109978
Taxonomy ID : 11036
Gene Strand (Orientation) : ?
Protein Name : PE2
Protein Length : 272
Protein Note : Alphavirus E3 glycoprotein; pfam01563

Protein Sequence : Show Sequence

>gi|229558446|gb|ACQ76875.1| PE2 [Venezuelan equine encephalitis virus]
LVTTMCLLANVTFPCAQPPICYDRKPAETLAMLSVNVDNPGYDELLEAAVKCPGRKRRSTEELFKEYKLT
RPYMARCIRCAVGSCHSPIAIEAVKSDGHDGYVRLQTSSQYGLDSSGNLKGRTMRYDMHGTIEEIPLHQV
SLHTSRPCHIVDGHGYFLLARCPAGDSITMEFKKDSVTHSCSVPYEVKFNPVGRELYTHPPEHGAEQACQ
VYAHDAQNRGAYVEMHLPGSEVDSSLVSLSGSSVTVTPPAGTSALVECECGGTKISETINTA

Molecule Role : Virmugen
Molecule Role Annotation : A VEE mutant with a PE2 cleavage signal mutation and a suppressor mutation in E1 is highly attenuated in mice and provides complete protection against challenge with wild type VEE (Davis et al., 1995).
Related Vaccine(s): VEE Virus PE2/E1 mutant vaccine

6. POLS_EEVVT Structural polyprotein (p130)

Gene Name : POLS_EEVVT Structural polyprotein (p130)
Sequence Strain (Species/Organism) : Venezuelan equine encephalitis virus (strain Trinidad donkey)
NCBI Protein GI : 109940317
Other Database IDs : GI:109940317;Swissprot:POLS_EEVVT; Swissprot:P09592;
Taxonomy ID : 11038
Gene Strand (Orientation) : ?
Protein Name : Structural polyprotein (p130)
Protein Length : 1254
Protein Annotation : Contains: Capsid protein (EC 3.4.21.-) (Coat protein) (C); p62 (E3/E2); E3 protein (Spike glycoprotein E3); E2 envelope glycoprotein (Spike glycoprotein E2); 6K protein; E1 envelope glycoprotein (Spike glycoprotein E1)

Protein Sequence : Show Sequence

>gi|109940317|sp|P09592|POLS_EEVVT Structural polyprotein (p130) [Contains: Capsid protein (Coat protein) (C); p62 (E3/E2); E3 protein (Spike glycoprotein E3); E2 envelope glycoprotein (Spike glycoprotein E2); 6K protein; E1 envelope glycoprotein (Spike glycoprotein E1)]
MFPFQPMYPMQPMPYRNPFAAPRRPWFPRTDPFLAMQVQELTRSMANLTFKQRRDAPPEGPSAKKPKKEA
SQKQKGGGQGKKKKNQGKKKAKTGPPNPKAQNGNKKKTNKKPGKRQRMVMKLESDKTFPIMLEGKINGYA
CVVGGKLFRPMHVEGKIDNDVLAALKTKKASKYDLEYADVPQNMRADTFKYTHEKPQGYYSWHHGAVQYE
NGRFTVPKGVGAKGDSGRPILDNQGRVVAIVLGGVNEGSRTALSVVMWNEKGVTVKYTPENCEQWSLVTT
MCLLANVTFPCAQPPICYDRKPAETLAMLSVNVDNPGYDELLEAAVKCPGRKRRSTEELFKEYKLTRPYM
ARCIRCAVGSCHSPIAIEAVKSDGHDGYVRLQTSSQYGLDSSGNLKGRTMRYDMHGTIKEIPLHQVSLHT
SRPCHIVDGHGYFLLARCPAGDSITMEFKKDSVTHSCSVPYEVKFNPVGRELYTHPPEHGVEQACQVYAH
DAQNRGAYVEMHLPGSEVDSSLVSLSGSSVTVTPPVGTSALVECECGGTKISETINKTKQFSQCTKKEQC
RAYRLQNDKWVYNSDKLPKAAGATLKGKLHVPFLLADGKCTVPLAPEPMITFGFRSVSLKLHPKNPTYLT
TRQLADEPHYTHELISEPAVRNFTVTEKGWEFVWGNHPPKRFWAQETAPGNPHGLPHEVITHYYHRYPMS
TILGLSICAAIATVSVAASTWLFCRSRVACLTPYRLTPNARIPFCLAVLCCARTARAETTWESLDHLWNN
NQQMFWIQLLIPLAALIVVTRLLRCVCCVVPFLVMAGAAAGAYEHATTMPSQAGISYNTIVNRAGYAPLP
ISITPTKIKLIPTVNLEYVTCHYKTGMDSPAIKCCGSQECTPTYRPDEQCKVFTGVYPFMWGGAYCFCDT
ENTQVSKAYVMKSDDCLADHAEAYKAHTASVQAFLNITVGEHSIVTTVYVNGETPVNFNGVKLTAGPLST
AWTPFDRKIVQYAGEIYNYDFPEYGAGQPGAFGDIQSRTVSSSDLYANTNLVLQRPKAGAIHVPYTQAPS
GFEQWKKDKAPSLKFTAPFGCEIYTNPIRAENCAVGSIPLAFDIPDALFTRVSETPTLSAAECTLNECVY
SSDFGGIATVKYSASKSGKCAVHVPSGTATLKEAAVELTEQGSATIHFSTANIHPEFRLQICTSYVTCKG
DCHPPKDHIVTHPQYHAQTFTAAVSKTAWTWLTSLLGGSAVIIIIGLVLATIVAMYVLTNQKHN

Related Vaccine(s): Defective adenovirus expressing VEEV E2 glycoprotein

7. TC-83

Gene Name : TC-83
Sequence Strain (Species/Organism) : Venezuelan equine encephalitis virus
NCBI Protein GI : 1375089
Other Database IDs : CDD:109979
CDD:190039
CDD:201519
CDD:201874
Taxonomy ID : 11036
Gene Strand (Orientation) : ?
Protein Name : structural polyprotein precursor
Protein Length : 1255
Protein Note : putative

Protein Sequence : Show Sequence

>gi|1375089|gb|AAB02517.1| structural polyprotein precursor [Venezuelan equine encephalitis virus]
MFPFQPMYPMQPMPYRNPFAAPRRPWFPRTDPFLAMQVQELTRSMANLTFKQRRDAPPEGPSAKKPKKEA
SQKQKGGGQGKKKKNQGKKKAKTGPPNPKAQNGNKKKTNKKPGKRQRMVMKLESDKTFPIMLEGKINGYA
CVVGGKLFRPMHVEGKIDNDVLAALKTKKASKYDLEYADVPQNMRADTFKYTHEKPQGYYSWHHGAVQYE
NGRFTVPKGVGAKGDSGRPILDNQGRVVAIVLGGVNEGSRTALSVVMWNEKGVTVKYTPENCEQWSLVTT
MCLLANVTFPCAQPPICYDRKPAETLAMLSVNVDNPGYDELLEAAVKCPGRKRRSTEELFNEYKLTRPYM
ARCIRCAVGSCHSPIAIEAVKSDGHDGYVRLQTSSQYGLDSSGNLKGRTMRYDMHGTIKEIPLHQVSLYT
SRPCHIVDGHGYFLLARCPAGDSITMEFKKDSVRHSCSVPYEVKFNPVGRELYTHPPEHGVEQACQVYAH
DAQNRGAYVEMHLPGSEVDSSLVSLSGSSVTVTPPDGTSALVECECGGTKISETINKTKQFSQCTKKEQC
RAYRLQNDKWVYNSDKLPKAAGATLKGKLHVPFLLADGKCTVPLAPEPMITFGFRSVSLKLHPKNPTYLI
TRQLADEPHYTHELISEPAVRNFTVTEKGWEFVWGNHPPKRFWAQETAPGNPHGLPHEVITHYYHRYPMS
TILGLSICAAIATVSVAASTWLFCRSRVACLTPYRLTPNARIPFCLAVLCCARTARAETTWESLDHLWNN
NQQMFWIQLLIPLAALIVVTRLLRCVCCVVPFLVMAGAAGAGAYEHATTMPSQAGISYNTIVNRAGYAPL
PISITPTKIKLIPTVNLEYVTCHYKTGMDSPAIKCCGSQECTPTYRPDEQCKVFTGVYPFMWGGAYCFCD
TENTQVSKAYVMKSDDCLADHAEAYKAHTASVQAFLNITVGEHSIVTTVYVNGETPVNFNGVKITAGPLS
TAWTPFDRKIVQYAGEIYNYDFPEYGAGQPGAFGDIQSRTVSSSDLYANTNLVLQRPKAGAIHVPYTQAP
SGFEQWKKDKAPSLKFTAPFGCEIYTNPIRAENCAVGSIPLAFDIPDALFTRVSETPTLSAAECTLNECV
YSSDFGGIATVKYSASKSGKCAVHVPSGTATLKEAAVELTEQGSATIHFSTANIHPEFRLQICTSYVTCK
GDCHPPKDHIVTHPQYHAQTFTAAVSKTAWTWLTSLLGGSAVIIIIGLVLATIVAMYVLTNQKHN

Molecule Role : Other
Related Vaccine(s): VEE virus recombinant vector vaccine RAd/VEEV , VEE virus recombinant vector vaccine RAd/VEEV#2 , VEE virus recombinant vector vaccine RAd/VEEV#3 encoding TC-83

8. V antigen from Y. pestis

Gene Name : V antigen from Y. pestis
Sequence Strain (Species/Organism) : Yersinia pestis biovar Microtus str. 91001
NCBI Gene ID : 2767532
NCBI Protein GI : 45478553
Locus Tag : YP_pCD52
Genbank Accession : AE017043
Protein Accession : NP_995380
Taxonomy ID : 229193
Plasmid No : pCD1
Gene Starting Position : 37959
Gene Ending Position : 38933
Gene Strand (Orientation) : +
Protein Name : V antigen LcrV
Protein pI : 5.66
Protein Weight : 35535.45
Protein Length : 324
Protein Note : Best Blastp hit = gi|7578513|gb|AAF64076.1|AF167309_1 (AF167309) V antigen [Yersinia pestis] ;. Contains Low calcium response V antigen (Interpro|IPR005413, Cellular Component: extracellular (GO:0005576), Biological Process: pathogenesis (GO:0009405))

DNA Sequence : Show Sequence

>gi|45478502:37959-38933 Yersinia pestis biovar Microtus str. 91001 plasmid pCD1, complete sequence
TATGATTAGAGCCTACGAACAAAACCCACAACATTTTATTGAGGATCTAGAAAAAGTTAGGGTGGAACAA
CTTACTGGTCATGGTTCTTCAGTTTTAGAAGAATTGGTTCAGTTAGTCAAAGATAAAAATATAGATATTT
CCATTAAATATGATCCCAGAAAAGATTCGGAGGTTTTTGCCAATAGAGTAATTACTGATGATATCGAATT
GCTCAAGAAAATCCTAGCTTATTTTCTACCCGAGGATGCCATTCTTAAAGGCGGTCATTATGACAACCAA
CTGCAAAATGGCATCAAGCGAGTAAAAGAGTTCCTTGAATCATCGCCGAATACACAATGGGAATTGCGGG
CGTTCATGGCAGTAATGCATTTCTCTTTAACCGCCGATCGTATCGATGATGATATTTTGAAAGTGATTGT
TGATTCAATGAATCATCATGGTGATGCCCGTAGCAAGTTGCGTGAAGAATTAGCTGAGCTTACCGCCGAA
TTAAAGATTTATTCAGTTATTCAAGCCGAAATTAATAAGCATCTGTCTAGTAGTGGCACCATAAATATCC
ATGATAAATCCATTAATCTCATGGATAAAAATTTATATGGTTATACAGATGAAGAGATTTTTAAAGCCAG
CGCAGAGTACAAAATTCTCGAGAAAATGCCTCAAACCACCATTCAGGTGGATGGGAGCGAGAAAAAAATA
GTCTCGATAAAGGACTTTCTTGGAAGTGAGAATAAAAGAACCGGGGCGTTGGGTAATCTGAAAAACTCAT
ACTCTTATAATAAAGATAATAATGAATTATCTCACTTTGCCACCACCTGCTCGGATAAGTCCAGGCCGCT
CAACGACTTGGTTAGCCAAAAAACAACTCAGCTGTCTGATATTACATCACGTTTTAATTCAGCTATTGAA
GCACTGAACCGTTTCATTCAGAAATATGATTCAGTGATGCAACGTCTGCTAGATGACACGAGGTA

Protein Sequence : Show Sequence

>gi|45478553|ref|NP_995380.1| V antigen LcrV [Yersinia pestis biovar Microtus str. 91001]
MIRAYEQNPQHFIEDLEKVRVEQLTGHGSSVLEELVQLVKDKNIDISIKYDPRKDSEVFANRVITDDIEL
LKKILAYFLPEDAILKGGHYDNQLQNGIKRVKEFLESSPNTQWELRAFMAVMHFSLTADRIDDDILKVIV
DSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDKSINLMDKNLYGYTDEEIFKAS
AEYKILEKMPQTTIQVDGSEKKIVSIKDFLGSENKRTGALGNLKNSYSYNKDNNELSHFATTCSDKSRPL
NDLVSQKTTQLSDITSRFNSAIEALNRFIQKYDSVMQRLLDDTR

Molecule Role : Protective antigen

III. Vaccine Information

1. Chimeric SIN/VEE Virus SIN-83

a. Vaccine Ontology ID:

VO_0004111

b. Type:

Recombinant vector vaccine

c. Antigen

All structural proteins derived from VEEV TC-83 (Paessler et al., 2003).

d. Preparation

The parental pToto1101 plasmid, encoding the SINV genome, and the pTC-83 plasmid, encoding the genome of VEEV TC-83, were obtained from Charles M. Rice (Rockefeller University, New York, N.Y.) and Richard Kinney (Centers for Disease Control, Fort Collins, Colo.), respectively. Fragments containing the SINV subgenomic promoter and the 5′ untranslated region (UTR) of the VEEV subgenomic RNA were generated by PCR amplification, cloned into the pRS2 plasmid for sequencing, and then used for generating the cDNA clone of the chimeric SIN-83S virus genome. The plasmid construct pSIN83 (Paessler et al., 2003) contained the promoter for SP6 RNA polymerase, followed by nucleotides (nt) 1 to 7601 of the SINV genome, nt 7536 to 11382 of VEEV TC-83 (with an additional C→T mutation of nt 7555), an AGGCCTTGGG sequence, and a 355-nt sequence containing the SINV 3′UTR (starting from nt 11394), poly(A) followed by an XhoI restriction site. Plasmids pZPC and pSH, containing infectious cDNAs of VEEV strains ZPC738 (subtype ID) and SH3 (subtype IC), respectively. The plasmids were purified and linearized. RNAs were synthesized and transfected into BHK-21 cells (Bredenbeek et al., 1993). Viruses were harvested after development of cytopathic effects, usually at 24 h following electroporation.

e. Virulence

None of the chimeric SIN/VEE viruses caused any detectable disease in adult mice after either intracerebral (i.c.) or subcutaneous (s.c.) inoculation, and all chimeras were more attenuated than the vaccine strain, VEEV TC83, in 6-day-old mice after i.c. infection (Paessler et al., 2003).

f. Description

The chimeric SIN/VEE viruses contain the replicative machinery from another alphavirus, Sindbis virus (SINV), and the structural genes from VEEV. The prototype chimeric virus SIN83 is capable of replicating in tissue culture and exhibits a safe and highly attenuated phenotype in mice and hamsters but induces a protective immune response against VEEV (Paessler et al., 2003). It is safe and efficacious in adult mice and hamsters and is potentially useful as VEEV vaccin. In addition, immunized animals provide a useful model for studying the mechanisms of the anti-VEEV neuroinflammatory response, leading to the reduction of viral titers in the CNS and survival of animals.

g. Mouse Response

Host Strain: NIH Swiss
Vaccination Protocol: Six-week-old, female NIH Swiss mice (12 per group) were inoculated on day 0 s.c. into the medial thigh with chimeric SIN/VEE viruses SIN-83. The live VEE TC-83 vaccine virus was used as control for comparison. One half of the animals (six per group) received an additional booster on day 28, which was performed in the same way as the initial immunization. All of the animals were bled on days 1, 2, and 3 and at 4 and 8 weeks after immunization. Serum samples from the first 3 days after immunization were tested for the presence of infectious virus by a plaque assay on BHK-21 cells (Paessler et al., 2003).
Persistence: To compare the virulence of the VEE TC-83 and SIN-83 viruses, 6-day-old mice were inoculated i.c. or s.c. with different doses of each virus ranging from 2 × 10^4 to 2 × 10^6 PFU. VEEV TC-83 was virulent for weanling mice regardless of the inoculation route. VEEV TC-83 was less pathogenic for weanling mice after s.c. inoculation (mortality rate, 10 to 20%). However, many of the surviving animals developed clinical disease and/or CNS sequelae. None of the SIN-83-inoculated animals had detectable clinical illness. Animals infected with VEEV TC-83 at the age of 6 days were highly inhibited in their growth compared to those infected with SIN-83 or compared to the noninfected control group of the same age (Paessler et al., 2003).
Immune Response: After 28 days, VEEV-specific neutralizing antibodies in the sera of SIN-83 and VEE TC-83 immunized groups. However, the titers in VEEV TC-83-immunized animals were higher. This can be explained by the higher replication levels of this virus in cell culture.
Side Effects: none
Challenge Protocol: Challenge studies to determine the protection against clinical encephalitis in the mouse model. Fifteen 6-week-old, female NIH Swiss mice were vaccinated with 5 x 10^5 PFU of each chimeric virus or PBS alone (control) in a total volume of 100 µl. After vaccination, each cohort of 15 animals was maintained for 8 weeks without any manipulation. Immunized animals were then challenged with VEEV subtype ID strain ZPC738 by using three different inoculation methods: (i) s.c. inoculation into the medial thigh with 10^6 PFU (roughly 10^6 50% lethal dose) per animal in 0.1 ml of PBS (five mice per group), (ii) i.c. inoculation into the left brain hemisphere with 2 x 10^5 PFU per animal in 20 µl of PBS (five mice per group), and (iii) intranasal (i.n.) inoculation with 2 x 10^5 PFU per animal in 20 µl of PBS (five mice per group). Mice were observed for clinical illness (for anorexia and/or paralysis) and/or death twice daily for a period of 2 months.

Challenge studies to determine protection against viral replication in the brain following i.c. or i.n. inoculation with ZPC738. After a group of mice was vaccinated, the first challenge with ZPC738 was performed using two different inoculation methods: (i) i.c. inoculation into the left brain hemisphere with 2 x 10^5 PFU in 20 µl of PBS and (ii) i.n. inoculation with 2 x 10^5 PFU in 20 µl of PBS. Two animals per group were euthanized on days 3, 7, and 28 after infection, and lungs, livers, spleens, kidneys, and brains were collected for viral titration or histological examinations. In addition, 10 animals per group were housed for 28 days after i.n. challenge with ZPC738, without any manipulation. On day 28, all animals from this group received the second i.n. dose of 2 x 10^5 PFU of ZPC738. Two animals per group were euthanized on days 3, 7, and 28 postchallenge, and organs were collected as described above.
Efficacy: All vaccinated mice were protected against lethal encephalitis following i.c., s.c., or intranasal (i.n.) challenge with the virulent VEEV ZPC738 strain (ZPC738). In spite of the absence of clinical encephalitis in vaccinated mice challenged with ZPC738 via i.n. or i.c. route, high levels of infectious challenge virus in the central nervous system (CNS) were regularly detected. However, infectious virus was undetectable in the brains of all immunized animals at 28 days after challenge (Paessler et al., 2003).

h. Hamster Response

Host Strain: golden hamster
Vaccination Protocol: Three 6-week-old female Syrian golden hamsters per viral strain were vaccinated s.c. in the right medial thigh with 5 x 105 PFU of SAAR/TRD, SIN/ZPC, SIN/TRD, or TC83 strain or PBS alone. Blood samples were obtained daily for the first 3 days after infection, and the animals were observed twice daily for 21 days. Serum viremia was determined by using a plaque assay on BHK-21 cells as previously described. The presence of neutralizing antibody in hamster serum samples was determined via a plaque reduction neutralization test, as described for the murine experiments
Side Effects: none
Challenge Protocol: Three weeks after vaccination, the hamsters were challenged s.c. in the medial thigh with ZPC738 at a dose of 106 PFU in a total volume of 100 µl of PBS (roughly 5 x 106 50% lethal dose). The animals were observed for 28 days, and deaths or cases of clinical illness were documented.
Efficacy: Hamsters vaccinated with chimeric SIN/VEE viruses were also protected against s.c. challenge with ZPC738.
Description: Six- to 8-week-old female Syrian golden hamsters (Mesocricetus auratus) were purchased from Harlan and acclimatized in the facility for a week prior to infection.

2. Defective adenovirus expressing VEEV E2 glycoprotein

a. Vaccine Ontology ID:

VO_0004116

b. Type:

Recombinant vector vaccine

c. Antigen

VEEV E2 glycoprotein

d. Gene Engineering of POLS_EEVVT Structural polyprotein (p130)

Type: Recombinant protein preparation
Description: Recombinant defective type 5 adenoviruses expressing the E3E26K structural genes of VEEV were prepared and examined (Phillpotts et al., 2005).
Detailed Gene Information: Click here.

e. Preparation

To make recombinant viruses, the core gene, the first three nucleotides at the 5′-end of the VEE E3 gene, the 3′-end of the 6K gene and the E1 gene were removed by restriction digestions. An initiation codon, the three nucleotides deleted from E3, the nucleotides removed from the 3′-end of the 6K gene and a termination codon were all added by ligation of oligonucleotide linkers. The E3–E2–6K fragment was then cloned into plasmid pMV101, derived from pMV100 by the addition of a unique Eco R1 restriction site at an Xba I site located downstream of the promoter sequence, to generate plasmid pVEEV. Plasmid pMV100, which contains the CMV major immediate early promoter and a polyadenylation signal. Three sites within the VEEV E2 glycoprotein were mutated by site-directed mutagenesis from the sequence found in the attenuated TC-83 strain to the sequence found in the virulent TrD strain. Each of the E3–E2–6K sequences from plasmids pVEEV, pVEEV#2 and pVEEV#3 were inserted into AHuman adenovirus type 5 (Ad5) dl309 to make recombinant adenoviruses. Ad5 dl309 has a deleted E1a gene (Jones et al., 1979) and is replication defective, requiring E1a complementation for replication. The E1a gene product may be supplied in trans by stably transfected 293 cells. A second deletion is found in the E3 region of this virus (Bett et al., 1995). The Ad5 vector is designed to enter mammalian cells and express proteins but it is defective for production of infectious progeny virus. All of the recombinant adenoviruses were purified by three rounds of terminal dilution in 293 cells cultured in 96-well plates. The VEEV inserts in the recombinant adenoviruses were characterised by sequencing (Phillpotts et al., 2005).

To prepare virulent virus stocks, suckling mice were infected intracerebrally of each supplied virus. Infected brains were harvested, prepared as tissue suspensions and clarified by centrifugation. The titre of each VEEV strain was determined by plaque formation under a carboxymethyl cellulose overlay in Vero cells (Phillpotts et al., 2005).

f. Description

Recombinant defective type 5 adenoviruses, expressing the E3E26K structural genes of VEEV were examined for their ability to protect mice against airborne challenge with virulent virus. After intranasal administration, good protection was achieved against the homologous serogroup 1A/B challenge virus (strain Trinidad donkey). There was less protection against enzootic serogroup II and III viruses, indicating that inclusion of more than one E3E26K sequence in a putative vaccine may be necessary. These studies confirm the potential of recombinant adenoviruses as vaccine vectors for VEEV and will inform the development of a live replicating adenovirus-based VEEV vaccine, deliverable by a mucosal route and suitable for use in humans (Phillpotts et al., 2005).

g. Mouse Response

Host Strain: Balb/c
Vaccination Protocol: Balb/c mice, 6–8 weeks old (Charles River Laboratories, UK) were immunised intranasally under halothane anaesthesia on days 0, 7 and 21 with 107.0 pfu of each recombinant adenovirus in 50 μl PBS.
Immune Response: The ability of serum to neutralise VEEV was examined in standard plaque reduction assays. Briefly, dilutions of serum and VEEV suspended in L15MM were mixed and incubated at 4 °C overnight. Residual infectious virus was estimated by plaque assay in Vero cells. A reduction in plaque numbers of equal to or greater than 50% was considered indicative of virus neutralisation.

Immunisation with RAd/VEEV#3 provides cross-protection against other epizootic and enzootic strains
Challenge Protocol: Seven days after the final immunisation, the animals were challenged via the airborne route by exposure for 20 min to a polydisperse aerosol generated by a Collison nebuliser (Phillpotts et al., 1997). Mice were contained loose within a closed box during airborne challenge. The virus dose was calculated by sampling the air in the box and assuming a respiratory minute volume for mice of 1.25 ml/g (Guyton, A.C., 1947). After challenge, mice were observed twice daily for clinical signs of infection (piloerection, hunching, inactivity, excitability and paralysis) by an observer who quantified these and was unaware of the treatment allocations. In accordance with UK Home Office requirements and as previously described, humane endpoints were used (Wright et al., 1998). These experiments therefore record the occurrence of severe disease rather than mortality. Even though it is rare for animals infected with virulent VEEV and showing signs of severe illness to survive, our use of humane endpoints should be considered when interpreting any virus dose expressed here as 50% lethal doses (LD50).
Efficacy: Optimal protection within the VEEV IA/B serogroup depends upon sequence homology
Description: Balb/c mice, 6-8 weeks old

3. Encephalomyelitis Eastern & Western & Venezuelan, Killed Virus Vaccine-Tetanus Toxoid (USDA: 4865.23)

a. Manufacturer:

Boehringer Ingelheim Vetmedica, Inc., Intervet Inc.

b. Vaccine Ontology ID:

VO_0002266

c. Type:

Inactivated or "killed" vaccine

d. Status:

Licensed

e. Location Licensed:

USA

f. Host Species for Licensed Use:

Horse

4. Encephalomyelitis Eastern & Western & Venezuelan, Killed Virus Vaccine-Tetanus Toxoid (USDA: 4865.27)

a. Manufacturer:

Boehringer Ingelheim Vetmedica, Inc.

b. Vaccine Ontology ID:

VO_0002268

c. Type:

Inactivated or "killed" vaccine

d. Status:

Licensed

e. Location Licensed:

USA

f. Host Species for Licensed Use:

Horse

5. Encephalomyelitis Eastern & Western & Venezuelan, Killed Virus Vaccine-Tetanus Toxoid (USDA: 4867.20)

a. Manufacturer:

Wyeth

b. Vaccine Ontology ID:

VO_0002269

c. Type:

Inactivated or "killed" vaccine

d. Status:

Licensed

e. Location Licensed:

USA

f. Host Species for Licensed Use:

Horse

6. Encephalomyelitis Eastern & Western & Venezuelan, Killed Virus Vaccine-Tetanus Toxoid (USDA: 4867.21)

a. Manufacturer:

Wyeth

b. Vaccine Ontology ID:

VO_0002270

c. Type:

Inactivated or "killed" vaccine

d. Status:

Licensed

e. Location Licensed:

USA

f. Host Species for Licensed Use:

Horse

7. Encephalomyelitis-Influenza Eastern & Western & Venezuelan, Killed Virus Vaccine-Tetanus Toxoid (USDA: 4875.23)

a. Manufacturer:

Wyeth

b. Vaccine Ontology ID:

VO_0002271

c. Type:

Inactivated or "killed" vaccine

d. Status:

Licensed

e. Location Licensed:

USA

f. Host Species for Licensed Use:

Horse

8. Encephalomyelitis-Influenza Eastern & Western & Venezuelan, Killed Virus Vaccine-Tetanus Toxoid (USDA: 4875.24)

a. Manufacturer:

Wyeth

b. Vaccine Ontology ID:

VO_0002272

c. Type:

Inactivated or "killed" vaccine

d. Status:

Licensed

e. Location Licensed:

USA

f. Host Species for Licensed Use:

Horse

9. Encephalomyelitis-Influenza Eastern & Western & Venezuelan, Killed Virus Vaccine-Tetanus Toxoid (USDA: 4875.A0)

a. Manufacturer:

Boehringer Ingelheim Vetmedica, Inc.

b. Vaccine Ontology ID:

VO_0002273

c. Type:

Inactivated or "killed" vaccine

d. Status:

Licensed

e. Location Licensed:

USA

f. Host Species for Licensed Use:

Horse

10. Encephalomyelitis-Influenza Eastern & Western & Venezuelan, Killed Virus Vaccine-Tetanus Toxoid (USDA: 4875.A1)

a. Manufacturer:

Intervet Inc.

b. Vaccine Ontology ID:

VO_0002274

c. Type:

Inactivated or "killed" vaccine

d. Status:

Licensed

e. Location Licensed:

USA

f. Host Species for Licensed Use:

Horse

11. Encephalomyelitis-Rhinopneumonitis-Influenza Eastern & Western & Venezuelan, Killed Virus Vaccine-Tetanus Toxoid (USDA: 4847.22)

a. Manufacturer:

Wyeth

b. Vaccine Ontology ID:

VO_0002258

c. Type:

Inactivated or "killed" vaccine

d. Status:

Licensed

e. Location Licensed:

USA

f. Host Species for Licensed Use:

Horse

12. Encephalomyelitis-Rhinopneumonitis-Influenza Eastern & Western & Venezuelan, Killed Virus Vaccine-Tetanus Toxoid (USDA: 4847.23)

a. Manufacturer:

Wyeth

b. Vaccine Ontology ID:

VO_0002259

c. Type:

Inactivated or "killed" vaccine

d. Status:

Licensed

e. Location Licensed:

USA

f. Host Species for Licensed Use:

Horse

13. Encephalomyelitis-Rhinopneumonitis-Influenza Eastern & Western & Venezuelan, Killed Virus Vaccine-Tetanus Toxoid (USDA: 4847.24)

a. Manufacturer:

Wyeth

b. Vaccine Ontology ID:

VO_0002260

c. Type:

Inactivated or "killed" vaccine

d. Status:

Licensed

e. Location Licensed:

USA

f. Host Species for Licensed Use:

Horse

14. Encephalomyelitis-Rhinopneumonitis-Influenza Eastern & Western & Venezuelan, Killed Virus Vaccine-Tetanus Toxoid (USDA: 4847.32)

a. Manufacturer:

Intervet Inc.

b. Vaccine Ontology ID:

VO_0002261

c. Type:

Inactivated or "killed" vaccine

d. Status:

Licensed

e. Location Licensed:

USA

f. Host Species for Licensed Use:

Horse

15. Encephalomyelitis-West Nile Virus Eastern & Western & Venezuelan, Killed Virus Vaccine (USDA: 14W5.23)

a. Manufacturer:

Wyeth

b. Vaccine Ontology ID:

VO_0002128

c. Type:

Inactivated or "killed" vaccine

d. Status:

Licensed

e. Location Licensed:

USA

f. Host Species for Licensed Use:

Horse

16. Encephalomyelitis-West Nile Virus Eastern & Western & Venezuelan, Killed Virus Vaccine-Tetanus Toxoid (USDA: 48W5.20)

a. Manufacturer:

Boehringer Ingelheim Vetmedica, Inc.

b. Vaccine Ontology ID:

VO_0002284

c. Type:

Inactivated or "killed" vaccine

d. Status:

Licensed

e. Location Licensed:

USA

f. Host Species for Licensed Use:

Horse

17. Encephalomyelitis-West Nile Virus Eastern & Western & Venezuelan, Killed Virus Vaccine-Tetanus Toxoid (USDA: 48W5.23)

a. Manufacturer:

Wyeth

b. Vaccine Ontology ID:

VO_0002287

c. Type:

Inactivated or "killed" vaccine

d. Status:

Licensed

e. Location Licensed:

USA

f. Host Species for Licensed Use:

Horse

18. Live attenuated V3526 virus

a. Vaccine Ontology ID:

VO_0004113

b. Type:

Live, attenuated vaccine

c. Preparation

The V3526 vaccine candidate was propagated by serial passage in MRC-5 cells to generate pilot-scale pre-master and master virus seed banks that were subsequently used to produce a bulk lot of virus vaccine. The VEEV TrD viruses used in the plaque reduction neutralizing antibody titer (PRNT) assays was produced by Southern Research Institute-Frederick (Frederick, MD) from a starting seed 15 passages from the original donkey virus isolate.

d. Description

V3526 is a live-attenuated virus derived by site-directed mutagenesis from a virulent clone of the Venezuelan equine encephalitis virus (VEEV) IA/B Trinidad donkey (TrD) strain. It is intended for human use in protection against Venezuelan equine encephalitis (VEE). Vaccinations with V3526, at doses as low as 102 pfu, were safe and efficacious in protecting horses against a virulent TrD virus challenge.

e. Horse Response

Vaccination Protocol: Horses were inoculated in the left shoulder with SC injection of V3526 vaccine or PCM administered SC. Clinical observations were recorded daily throughout the immunization phase and for 14 days PC. Blood and data were collected from all horses throughout the course of the study with Day 0 samples taken pre-vaccination and designated as the day of vaccination. Day 28 PV blood was collected prior to administration of the virus challenge. Blood samples for virus isolation were collected daily on Days 0–10 PV, Days 14 and 21 PV, and daily PC (Days 28–38 PV) (or until euthanasia) and on Day 42 PV for surviving horses.
Challenge Protocol: Horses in all three trials were challenged on Day 28 PV with a 1 mL SC injection in the left shoulder with either 104 pfu TrD or 104 pfu of 64A99, a challenge dose comparable to that used in previous vaccine protection studies in mice [8]. Horses were monitored for signs of disease for 14 days PC.

Blood was evaluated for viremia (via plaque assay) and sera for VEEV neutralizing antibodies. Blood for hematology analysis was collected once daily from Days −1 to 10 PV, and from Days 27 to 38 PV. Blood was analyzed using a QBC-V hematology analyzer (Clay Adams, Inc.) for hematocrit, red blood cells, platelet and total white blood cell (WBC) count, as well as percentage and absolute numbers of granulocytes and mononuclear (lymphocyte and monocyte) cells (PBMCs).
Efficacy: None of the horses that received V3526 vaccine or PCM showed clinical signs of disease through the entire pre-challenge period. None of the horses developed a viremia after V3526 inoculation. All 25 horses vaccinated with V3526 vaccine, regardless of dose, survived challenge with either TrD or 64A99. In contrast, all five unvaccinated control horses developed severe disease, including anorexia, fever and malaise, beginning 1–2 days after TrD challenge.
Description: These studies utilized 33 mixed breed horses, mostly quarter horse stock, with nearly equal numbers of males and females, and in the age range of 3–14 years (as estimated from dental examination). All horses tested negative for pre-existing virus neutralizing antibodies to EEEV, WEEV and VEEV by plaque reduction neutralization (PRN) assays.

19. Live attenuated vaccine TC-83

a. Vaccine Ontology ID:

VO_0004105

b. Type:

Live, attenuated vaccine

c. Antigen

d. Preparation

Stocks of TC-83 were prepared by propagation from a vial of vaccine for human use (National Drug Company, Philadelphia PA, Lot 4, run 2). Virulent VEEV strains were prepared as suckling mouse brain suspensions by standard methods. In vitro virus propagation and plaque assays were performed in BHK21 cells, grown in Glasgow minimal essential medium. A hyperimmune rabbit antiserum raised against TC-83 virus was kindly provided by T. Webber, DET, at CBD, Porton Down.

e. Virulence

none

f. Description

TC-83 has proven safe and effective for immunisation of horses (Eddy et al., 1972; Walton et al., 1972)and has US Food and Drug Administration Investigational New Drug status for use in Humans (IND No. 142). Vaccination with TC-83 virus produced solid protection against subcutaneous challenge with Venezuelan equine encephalitis (VEEV) viruses from homologous and heterologous serogroups, but breakthrough infection and disease occurred after airborne challenge.

g. Mouse Response

Host Strain: outbred Porton TO
Vaccination Protocol: Subcutaneous (s.c.) inoculations of TC-83 virus in L15 MM, L15 MM alone, or virulent VEEV in L15 MM were delivered into the scruff of the neck in a volume of 100 μl. The number of s.c. LD50/ml was determined by titration using five mice per dilution.
Challenge Protocol: Infection by the airborne route was achieved using a specially constructed small animal exposure box(Phillpotts et al., 1997). Briefly, mice were exposed loose in a box of 80 l capacity, vented through a HEPA filter, to a virus-containing aerosol produced using a Collison nebuliser (8 l/min). The virus suspending fluid was L15 MM with 2% trehalose and the exposure time was 20 min. The air in the box was sampled with a glass impinger running at 1 l/min and the quantity of virus present in the Collison spray reservoir at the end of each run was determined by back-titration. A titration experiment was performed for each virus (five mice per dilution) in order to calculate the number of LD50 delivered by a given dilution of virus in the spray. As there was a linear relationship between the quantity of virus in the Collison reservoir and the impinger, in subsequent experiments the dose of virus delivered was calculated from titration of the impinger contents. Data for each virus at each time point were from a single exposure.
Efficacy: Vaccination with TC-83 virus produced solid protection against subcutaneous challenge with Venezuelan equine encephalitis (VEEV) viruses from homologous and heterologous serogroups, but breakthrough infection and disease occurred after airborne challenge.
Description: Infection by the airborne route was achieved using a specially constructed small animal exposure box(Phillpotts et al., 1997).

20. Live attenuated VEE vaccines

a. Vaccine Ontology ID:

VO_0004112

b. Type:

Live, attenuated vaccine

c. Preparation

The generation of isogenic molecular clones and viral stocks for this study was previously described. Briefly, a full-length cDNA clone of the wild-type Trinidad donkey strain of VEE (TrD), pV3000 (Davis et al., 1989), served as the template. VEEV clones with either single or multiple mutations were constructed for site-directed mutagenesis of a M13 subclone of the glycoprotein genes in pV3000. Infectious VEEV RNA, transcribed in vitro from these clones (e.g. pV3519), was used to produce virus (e.g. termed V3519) by transfection of baby hamster kidney (BHK) cells (Davis et al., 1991). Virus stocks tested in these studies were obtained directly from transfected BHK culture supernatant fluids and were used after appropriate dilution without further passage. Parent V3000 virus was passaged twice in BHK cells after collection from transfection supernatant fluids.

d. Description

Molecular clones of vaccine candidates were constructed by inserting either three independently attenuating mutations or a PE2 cleavage-signal mutation with a second-site resuscitating mutation into full-length cDNA clones. Vaccine candidate viruses were recovered through DNA transcription and RNA transfection of cultured cells, and assessed in rodent and non-human primate models. Based on results from this assessment, one of the PE2 cleavage-signal mutants, V3526, was determined to be the best vaccine candidate for further evaluation for human use.

e. Mouse Response

Host Strain: C57BL/6
Vaccination Protocol: Female C57BL/6 mice (8–10 weeks) were inoculated s.c. with 0.2 ml of cell culture medium containing either no virus, or plaque forming units (pfu) of the virulent V3000 virus or one of the mutant viral strains. Groups of animals inoculated with either a single human dose of TC-83 or three human doses of C-84 on days 0, 7 and 28 were used as comparisons. The degree of attenuation of the viral strains was assessed during the 14-day observation period after inoculation. On day 49 after inoculation, surviving mice were bled from the retro-orbital sinus under methoxyflurane (Pitman-Moore, Mundelein, IL) anesthesia.
Challenge Protocol: On day 55 after the primary inoculation, animals were challenged with a calculated dose of 105 pfu of V3000 or 104 pfu of TrD by aerosol exposure or by intraperitoneal inoculation. For aerosol challenge, animals were exposed for 10 min to an infectious aerosol generated by a Collison nebulizer within a Plexiglass chamber contained within a Class III biological safety cabinet located in a Biosafety Level 3 laboratory. Viral doses delivered by aerosol were calculated by standard procedures(Hart et al., 1997). Protection was assessed by monitoring animals for 28 days post-infection. Additional groups of animals were inoculated with selected viruses (V3526 or V3528) or TC-83 by aerosol and then challenge by aerosol with V3000 on day 55 post-inoculation to evaluate the ability of these viruses to induce mucosal immunity and protect against aerosol challenge.
Efficacy: All single mutants tested in mice were fully attenuated and induced protective immune responses (data not shown).
Description: female 8-10 weeks

f. Hamster Response

Host Strain: Syrian
Vaccination Protocol: Female Syrian (7–9 weeks) were inoculated s.c. with 0.2 ml of cell culture medium containing either no virus, or plaque forming units (pfu) of the virulent V3000 virus or one of the mutant viral strains. Groups of animals inoculated with either a single human dose of TC-83 or three human doses of C-84 on days 0, 7 and 28 were used as comparisons. The degree of attenuation of the viral strains was assessed during the 14-day observation period after inoculation. On day 49 after inoculation, surviving hamsters were bled by cardiac puncture under tiletamine-zolazepam (50 mg/kg, Aveco Co., Inc., Fort Dodge, IA) anesthesia.
Challenge Protocol: On day 55 after the primary inoculation, animals were challenged with a calculated dose of 105 pfu of V3000 or 104 pfu of TrD by aerosol exposure or by intraperitoneal inoculation. For aerosol challenge, animals were exposed for 10 min to an infectious aerosol generated by a Collison nebulizer within a Plexiglass chamber contained within a Class III biological safety cabinet located in a Biosafety Level 3 laboratory. Viral doses delivered by aerosol were calculated by standard procedures(Hart et al., 1997). Protection was assessed by monitoring animals for 28 days post-infection. Additional groups of animals were inoculated with selected viruses (V3526 or V3528) or TC-83 by aerosol and then challenge by aerosol with V3000 on day 55 post-inoculation to evaluate the ability of these viruses to induce mucosal immunity and protect against aerosol challenge.
Efficacy: All single mutants, when tested in hamsters, ranged from fully virulent to partially attenuated. In general, hamsters that survived did generate protective immunity against aerosol challenge.
Description: female 7-9 weeks

g. Monkey Response

Host Strain: Macaca fascicularis
Vaccination Protocol: The non-human primate model monkey used to test the safety and efficacy of VEEV vaccines was previously described (Pratt et al., 2003). Briefly, 30 healthy cynomolgus macaques were s.c. implanted with radiotelemetry devices to monitor body temperatures. During the pre-vaccination and pre-challenge periods (day −10 to day 0) and the 21 days after vaccination and challenge, body temperatures were recorded every 15 min. An autoregressive integrated moving average model (BMDP Statistics Software 1992) for each monkey was developed using the averaged hourly body temperature data over a baseline-training period (day −10 to day −3) and was used to forecast normal body temperature values during the vaccination and challenge time periods. Significant temperature elevations were used to compute fever duration (number of hours or days of significant temperature elevation) and fever-hours (sum of the significant temperature elevations).

Monkeys were randomly divided into six groups (N=5) and each monkey received a single s.c. 0.5 ml dose of a vaccine candidate (V3524, V3526 or V3528), TC-83, V3000 or virus-free cell culture medium. On day 35 or 36 after inoculation, bronchial lavage and blood samples were collected for N antibody titrations.
Side Effects: fever for V3524, V3526, V3528, TC-83. Four days of significant fever for wild type V3000
Challenge Protocol: On days 42 or 43, monkeys were anaesthetized and exposed for to an infectious aerosol of V3000. Monkeys were bled daily for 6 days after both immunization and challenge to monitor viremias and lymphocyte counts. On day 14 post-challenge, serum was collected for N antibody titrations. Virus dosages, viremias, and N antibody titers were determined in similar manner to those for the rodent studies. Statistical evaluation of the groups of monkeys was made using analysis of variance followed by multiple comparisons using the Tukey studentized range test (SAS ver. 6.10, Cary, NC).
Efficacy: Monkeys vaccinated with V3526 or V3528 were well protected against aerosol challenge with few to no signs of fever, lymphopenia, or viremia—similar to the group of monkeys previously inoculated with V3000. The group of monkeys vaccinated with TC-83 was also in this category, but one monkey that did not have pre-challenge N antibody titers did develop fever responses similar to the mock-inoculated monkeys. Unlike the groups of monkeys inoculated with V3526, V3528, TC-83, or V3000, the group of monkeys inoculated with V3524 was not as well protected against aerosol challenge with V3000 and was in a distinct fever grouping. It suggested a higher degree of infection and viral replication in these monkeys. Additionally, viremia was present in one V3524-inoculated monkey.
Description: 30 healthy cynomolgus macaques (Macaca fascicularis, 4.2–6.7 kg), screened negative by ELISA for previous exposure to alphaviruses

21. live TC-83 VEE Vaccine with DHEA

a. Vaccine Ontology ID:

VO_0004261

b. Type:

Live, attenuated vaccine

c. Status:

Research

d. Adjuvant:

VO ID: VO_0001340
Description: DHEA (Sigma Chemical Co., St. Louis, MO, U.S.A.) was dissolved in 50% ethanol. Mice were injected with DHEA (10 mg/Kg), in 0.3 mL, subcutaneously (s.c.), 4 hours before vaccination (Negrette et al., 2001).

e. Immunization Route

Intraperitoneal injection (i.p.)

f. Mouse Response

Host Strain: NMRI-IVIC
Vaccination Protocol: Mice were immunized intraperitoneally (i.p.) with 0.05 mL of live TC-83 VEE virus suspension, containing 1.7 x 106 PFU/mL, in 0.4% BABS. Virus stock from IVIC was replicated in VERO cell culture and contained 106.78 LD50. DHEA (Sigma Chemical Co., St. Louis, MO, U.S.A.) was dissolved in 50% ethanol. Mice were injected with DHEA (10 mg/Kg), in 0.3 mL, subcutaneously (s.c.), 4 hours before vaccination. Control mice were injected with the diluent (Negrette et al., 2001).
Immune Response: In mice treated with a single dose of DHEA, a significant increase (p<0.01) of antibody titers (Control = 1:1.680 vs. DHEA treated group = 1:4.320) was detected at week 2 after vaccination with TC-83 virus (Negrette et al., 2001).
Challenge Protocol: Live VEE virus (10 LD50) was injected in mice treated with DHEA, 21 days after immunization, and 2 to 5 days later they were sacrificed to determine viral titers in serum and brain (Negrette et al., 2001).
Efficacy: MIce immunized with the TC-83 virus and DHEA had reduced viremia in the blood and brain after challenge (Negrette et al., 2001).

22. Recombinant RNA replicons from attenuated VEE virus

a. Vaccine Ontology ID:

VO_0004109

b. Type:

Recombinant vector vaccine

c. Preparation

A plasmid encoding the VEE replicon vector was described previously (Davis et al., 1996; Pushko et al., 1997). The Ebola NP and GP genes from the Mayinga strain of Ebola virus were derived from pSP64- and pGEM3Zf(ÿ)-based plasmids (Sanchez et al., 1993; Sanchez et al., 1989). The fragments containing the NP and GP genes, respectively, were subcloned into a shuttle vector(Davis et al., 1996; Grieder et al., 1995). From the shuttle vector, NP or GP genes were transferred the replicon clone, resulting in plasmids encoding the NP or GP gene in place of the VEE structural protein genes.

Transcripts of the replicon were pooled and cotransfected as described previously (Pushko et al., 1997). Transfected cells were incubated and harvested. Culture supernatants were clarified by centrifugation and VRP particles were concentrated and partially purified by centrifugation. VRP titers were determined as immunofluorescent foci after infection of BHK cells in eight-well chamber slides as previously described (Pushko et al., 1997; Pifat et al., 1988). Positive cells were counted, and the titers of the VRP preparations were calculated. Production and titration of Lassa N±VRP were described previously (Pushko et al., 1997). For expression assays, BHK cells were infected with NP- or GP±VRP, or cotransfected with replicon and helper RNAs. Cells were incubated before harvesting. Polypeptides were separated and Western blotting was carried out. VEE proteins in western blots were detected with sera from guinea pigs immunized with the live-attenuated TC-83 vaccine.

d. Description

RNA replicons derived from an attenuated strain of Venezuelan equine encephalitis virus (VEE), an alphavirus, were configured as candidate vaccines for Ebola hemorrhagic fever. The Ebola nucleoprotein (NP) or glycoprotein (GP) genes were introduced into the VEE RNA downstream from the VEE 26S promoter in place of the VEE structural protein genes. The resulting recombinant replicons, expressing the NP or GP genes, were packaged into VEE replicon particles (NP–VRP and GP–VRP, respectively). The immunogenicity of NP–VRP and GP–VRP and their ability to protect against lethal Ebola infection were evaluated in BALB/c mice and in two strains of guinea pigs. The GP–VRP alone, or in combination with NP–VRP, protected both strains of guinea pigs and BALB/c mice, while immunization with NP–VRP alone protected BALB/c mice, but neither strain of guinea pig. Passive transfer of sera from VRP-immunized animals did not confer protection against lethal challenge. However, the complete protection achieved with active immunization with VRP, as well as the unique characteristics of the VEE replicon vector, warrant further testing of the safety and efficacy of NP–VRP and GP–VRP in primates as candidate vaccines against Ebola hemorrhagic fever.

e. Mouse Response

Host Strain: BALB/c
Vaccination Protocol: VRP were diluted and administered to BALB/c mice. Groups of mice were inoculated subcutaneously (s.c.).
Side Effects: none
Challenge Protocol: Challenge was carried out 4 weeks after final immunization with VRP. Mice were challeged i.p. with mouse-adapted Ebola virus. Animals wre observed daily for 60 days and morbidity and survival were recorded.
Efficacy: The GP-VRP alone, or in combination with NP-VRP, protected BALB/c mice. Immunization with NP-VRP alone protected BALB/c mice. Passive transfer of sera from VRP-immunized animals did not confer protection against lethal challenge.

f. Guinea pig Response

Host Strain: strain 2 or strain 13
Vaccination Protocol: VRP were diluted and administered to BALB/c mice. Groups of guinea pigs were inoculated subcutaneously (s.c.).
Side Effects: none
Challenge Protocol: Challenge was carried out 4 weeks after final immunization with VRP. Guinea pigs were challeged s.c. with guinea pig-adapted Ebola virus. Animals wre observed daily for 60 days and morbidity and survival were recorded.
Efficacy: The GP-VRP alone, or in combination with NP-VRP, protected both strains of guinea pigs. Immunization with NP-VRP alone protected neither strain of guinea pigs. Passive transfer of sera from VRP-immunized animals did not confer protection against lethal challenge.

23. VEE virus complex-specific monoclonal antibody

a. Vaccine Ontology ID:

VO_0004104

b. Type:

Monoclonal antibody

c. Antigen

No VEE virus antigens were used for this vaccination approach. The monoclonal antibodies are specifically designed against VEEV surface glycoproteins E1 and E2.

d. Preparation

The monoclonal antibodies (MAB) are: (1). the VEEV surface glycoprotein E2-specific monoclonal antibodies 1A4A-1; (2). the VEEV surface glycoprotein E2-specific monoclonal antibodies 1A3B-7; (3) the glycoprotein E1-specific MAB 3B2A-9; (4) 4. the E1-specific MAB MH2. All MAB were produced and purified by protein G affinity chromatography as previously described (Phillpotts et al., 2002).

Strains of VEEV from serogroups IA/B (Trinidad donkey; TrD), IC (P676), ID (3880), IE (Menall), IF (78v-3531), II (Fe37c), IIIA (Mucambo BeAn8), IV (Pixuna BeAn356445), V (Cabassou CaAr508) and IV (AG80-663) are used.
For the preparation of virus stocks, suckling mice were infected intracerebrally with a 1/1000 dilution of each virus. Infected brains were harvested at 24 h, prepared as 10% tissue suspensions in L15MM and clarified by centrifugation at 10,000 × g for 15 min. Alternatively viruses were propagated in Vero cell monolayers inoculated with an m.o.i. of 0.01 and harvested when 50–75% of the cell sheet showed evidence of virus cytopathic effect (CPE). The titre of each VEEV strain was determined by plaque formation under a carboxymethyl cellulose overlay in BHK21 cells.

e. Virulence

Not virulent

f. Description

Using a murine model to study monoclonal antibody (MAB) a VEEV complex-specific, glycoprotein E2-binding MAB was identified, able to protect against disease induced by exposure to aerosolised VEEV from serogroups I, II and IIIA (mouse-virulent strains). There was no synergy in protection between anti-E1 and anti-E2 MAB. Assays of MAB virus neutralising activity in a homologous (mouse fibroblast) cell line suggested that neutralisation played a significant role in protection in addition to the previously reported mechanism of Fc receptor-binding [Mathews et al., 1985. J. Virol. 55, 594–600]. Development of an analogous human MAB with identical VEEV epitope specificity may be informed and monitored by reference to these properties (Phillpotts, 2006).

g. Mouse Response

Host Strain: Balb/c mice
Vaccination Protocol: Balb/c mice, 3–4 (s.c. challenge) or 6–8 (airborne challenge) weeks old (Charles River Laboratories, UK) were passively immunized intraperitoneally (i.p.) 24 h before exposure to virulent virus with MAB diluted in PBS.
Side Effects: No side effect
Challenge Protocol: The challenge route was either s.c. (100 μl virus-containing suspension in PBS) or via the airborne route, by exposure for 20 min to a poly disperse aerosol generated by a Collison nebuliser (Phillpotts et al., 1997). Mice were contained loose within a closed box during airborne challenge. The virus dose was calculated by sampling the air in the box and assuming a respiratory minute volume for mice of 1.25 ml/g (Guyton, A.C., 1947). After challenge, mice were observed twice daily for clinical signs of infection (piloerection, hunching, inactivity, excitability and paralysis) by an observer who quantified these and was unaware of the treatment allocations. These clinical signs are typical of VEEV infection and occurred in all mice challenged with VEEV.
In previous studies (Phillpotts et al., 2002) their association with VEEV infection as the cause of death has been confirmed by the isolation of virus from brain and other internal organs. In accordance with UK Home Office requirements and as previously described, humane endpoints were used (Wright et al., 1998). These experiments therefore record the occurrence of severe disease rather than mortality. Even though it is rare for animals infected with virulent VEEV and showing signs of severe illness to survive, our use of humane endpoints should be considered when interpreting any virus dose expressed here as 50% lethal doses (LD50).
Efficacy: MAB MH2, 3B2A-9 and 1A3B-7 may be broadly reactive and potentially able to protect against a range of VEEV strains. There was also the possibility that the combination of E1- and E2-specific MAB could lead to synergy in protection. MAB 1A4A-1 while not broadly reactive, was able to bind strongly to, neutralise and protect against challenge with some VEEV strains. It was therefore included in these experiments as a positive control.
Description: The challenge route was either s.c. (100 μl virus-containing suspension in PBS) or via the airborne route, by exposure for 2

24. VEE virus DNA vaccine encoding 26S

a. Vaccine Ontology ID:

VO_0004488

b. Type:

DNA vaccine

c. Status:

Research

d. Host Species as Laboratory Animal Model:

Mouse

e. Gene Engineering of 26S mRNA

Type: DNA vaccine construction
Description: Vector pWRG7077 expressed VEEV 26S gene (Riemenschneider et al., 2003).
Detailed Gene Information: Click here.

f. Vector:

pWRG7077 (Riemenschneider et al., 2003)

g. Immunization Route

Intramuscular injection (i.m.)

h. Mouse Response

Vaccine Immune Response Type: VO_0000286
Efficacy: DNA vaccines to four dissimilar pathogens that are known biowarfare agents, Bacillus anthracis, Ebola (EBOV), Marburg (MARV), and Venezuelan equine encephalitis virus (VEEV), can elicit protective immunity in relevant animal models. In addition, a combination of all four vaccines is shown to be equally as effective as the individual vaccines for eliciting immune responses in a single animal species (Riemenschneider et al., 2003).

25. VEE virus DNA vaccine pSTU-TRDF encoding VEEV E3–E2–6K

a. Vaccine Ontology ID:

VO_0004490

b. Type:

DNA vaccine

c. Status:

Research

d. Host Species as Laboratory Animal Model:

Mouse

e. Gene Engineering of C-E3-E2-E1-6K

Type: DNA vaccine construction
Description:
Detailed Gene Information: Click here.

f. Vector:

pJW4304 prime, adenovirus-based vector boost (Perkins et al., 2006)

g. Immunization Route

Intramuscular injection (i.m.)

h. Mouse Response

Vaccine Immune Response Type: VO_0000286
Efficacy: Particle-mediated epidermal delivery of a DNA vaccine encoding the E2 glycoprotein of VEEV can be boosted with a mucosally-delivered Ad-based vaccine encoding the same E2 glycoprotein, resulting in a significant increase in protection against airborne VEEV (Perkins et al., 2006).

26. VEE virus DNA vaccine VEEV IA/B parent encoding structural genes

a. Vaccine Ontology ID:

VO_0004421

b. Type:

DNA vaccine

c. Status:

Research

d. Host Species as Laboratory Animal Model:

Mouse

e. Antigen

VEEV subtype IA/B capsid protein (C) and envelope glycoproteins (E1 and E2) (Dupuy et al., 2009)

f. Gene Engineering of E1 glycoprotein

Type: DNA vaccine construction
Description:
Detailed Gene Information: Click here.

g. Gene Engineering of E2 envelope protein

Type: DNA vaccine construction
Description:
Detailed Gene Information: Click here.

h. Vector:

pES (Dupuy et al., 2009)

i. Immunization Route

Intradermal injection (i.d.)

j. Mouse Response

Vaccine Immune Response Type: VO_0000286
Immune Response: The vaccine was able to induce increased total anti-VEEV IA/B antibody responses and increased neutralizing antibody responses (Dupuy et al., 2009).
Efficacy: The VEEV IA/B parent construct protected 80% of the mice from the lethal challenge, which is the same level of protection elicited by this DNA vaccine in a previous study. As in the previous study, mice vaccinated with the VEEV IA/B parent displayed signs of illness after challenge including ruffled fur, loss of appetite, and inactivity; however, the surviving mice completely recovered before the end of the 28 day post-challenge observation period (Dupuy et al., 2009).

27. VEE Virus PE2/E1 mutant vaccine

a. Type:

Live, attenuated vaccine

b. Status:

Research

c. Host Species as Laboratory Animal Model:

Mouse

d. Gene Engineering of E1 glycoprotein

Type: Gene mutation
Description: This PE2/E1 mutant is from Venezuelan equine encephalitis virus (Davis et al., 1995).
Detailed Gene Information: Click here.

e. Gene Engineering of PE2

Type: Gene mutation
Description: This PE2/E1 mutant is from Venezuelan equine encephalitis virus (Davis et al., 1995).
Detailed Gene Information: Click here.

f. Immunization Route

intranasal immunization

g. Mouse Response

Persistence: A PE2/E1 mutant is highly attenuated in mice (Davis et al., 1995).
Efficacy: A PE2/E1 mutant induces complete protection in mice from challenge with wild type VEE virus (Davis et al., 1995).

28. VEE virus recombinant vector vaccine RAd/VEEV

a. Vaccine Ontology ID:

VO_0004422

b. Type:

Recombinant vector vaccine

c. Status:

Research

d. Host Species as Laboratory Animal Model:

Mouse

e. Antigen

E3–E2–6K from VEEV strain TC-83 (Phillpotts et al., 2005)

f. Vector:

recombinant adenovirus (Phillpotts et al., 2005)

g. Immunization Route

Intramuscular injection (i.m.)

h. Mouse Response

Vaccine Immune Response Type: VO_0000286
Immune Response: Intranasal immunisation of mice with this recombinant virus produced a vigorous antibody response, with local IgA also detectable in respiratory secretions accompanied by highly significant protection against airborne VEEV challenge (Phillpotts et al., 2005).
Efficacy: This vaccine provided protection against low dose challenge (73 LD50); 4 out of 6 challenged mice survived (Phillpotts et al., 2005).

29. VEE virus recombinant vector vaccine RAd/VEEV#2

a. Vaccine Ontology ID:

VO_0004423

b. Type:

Recombinant vector vaccine

c. Status:

Research

d. Host Species as Laboratory Animal Model:

Mouse

e. Antigen

E3-E2-6K from VEEV TC-83 with two mutations in the major neutralisation site of E. (Phillpotts et al., 2005)

f. Vector:

recombinant adenovirus (Phillpotts et al., 2005)

g. Immunization Route

Intramuscular injection (i.m.)

h. Mouse Response

Vaccine Immune Response Type: VO_0000286
Immune Response: Intranasal immunisation of mice with this recombinant virus produced a vigorous antibody response, with local IgA also detectable in respiratory secretions accompanied by highly significant protection against airborne VEEV challenge (Phillpotts et al., 2005).
Efficacy: This vaccine provided protection against low dose challenge (73 LD50); 4 out of 6 challenged mice survived (Phillpotts et al., 2005).

30. VEE virus recombinant vector vaccine RAd/VEEV#3 encoding TC-83

a. Vaccine Ontology ID:

VO_0004424

b. Type:

Recombinant vector vaccine

c. Status:

Research

d. Host Species as Laboratory Animal Model:

Mouse

e. Antigen

E3-E2-6K from VEEV TC-83 with two mutations in the major neutralisation site of E2 and an additional mutation at the N-terminus of E2 (Phillpotts et al., 2005)

f. Vector:

recombinant adenovirus (Phillpotts et al., 2005)

g. Immunization Route

Intramuscular injection (i.m.)

h. Mouse Response

Vaccine Immune Response Type: VO_0000286
Immune Response: Intranasal immunisation of mice with this recombinant virus produced a vigorous antibody response, with local IgA also detectable in respiratory secretions accompanied by highly significant protection against airborne VEEV challenge (Phillpotts et al., 2005).
Efficacy: This vaccine provided protection against low and intermediate dose challenge (73 LD50 and 640 LD50, respectively); 5 out of 6 mice survived low dose challenge and 3 out of 5 mice survived intermediate dose challenge (Phillpotts et al., 2005).

IV. References

1. Agapov et al., 1994: Agapov EV, Razumov IA, Frolov IV, Kolykhalov AA, Netesov SV, Loktev VB. Localization of four antigenic sites involved in Venezuelan equine encephalomyelitis virus protection. Archives of virology. 1994; 139(1-2); 173-181. [PubMed: 7529989].

2. Bennett et al., 1999: Bennett AM, Phillpotts RJ, Perkins SD, Jacobs SC, Williamson ED. Gene gun mediated vaccination is superior to manual delivery for immunisation with DNA vaccines expressing protective antigens from Yersinia pestis or Venezuelan Equine Encephalitis virus. Vaccine. 1999; 18(7-8); 588-596. [PubMed: 10547416].

3. Bennett et al., 2000: Bennett AM, Elvin SJ, Wright AJ, Jones SM, Phillpotts RJ. An immunological profile of Balb/c mice protected from airborne challenge following vaccination with a live attenuated Venezuelan equine encephalitis virus vaccine. Vaccine. 2000 Sep 15; 19(2-3); 337-47. [PubMed: 10930689].

4. Bett et al., 1995: Bett AJ, Krougliak V, Graham FL. DNA sequence of the deletion/insertion in early region 3 of Ad5 dl309. Virus research. 1995 Nov; 39(1); 75-82. [PubMed: 8607286].

5. BMDP Statistics Software 1992: . BMDP Statistics Software. 467. BMDP Statistics Software Release 7. 1992. , .

6. Bray et al., 1999: Bray M, Davis K, Geisbert T, Schmaljohn C, Huggins J. A mouse model for evaluation of prophylaxis and therapy of Ebola hemorrhagic fever. The Journal of infectious diseases. 1999 Feb; 179 Suppl 1; S248-58. [PubMed: 9988191].

7. Bredenbeek et al., 1993: Bredenbeek PJ, Frolov I, Rice CM, Schlesinger S. Sindbis virus expression vectors: packaging of RNA replicons by using defective helper RNAs. Journal of virology. 1993 Nov; 67(11); 6439-46. [PubMed: 8411346 ].

8. Connolly et al., 1999: Connolly BM, Steele KE, Davis KJ, Geisbert TW, Kell WM, Jaax NK, Jahrling PB. Pathogenesis of experimental Ebola virus infection in guinea pigs. The Journal of infectious diseases. 1999 Feb; 179 Suppl 1; S203-17. [PubMed: 9988186].

9. Davis et al., 1989: Davis NL, Willis LV, Smith JF, Johnston RE. In vitro synthesis of infectious venezuelan equine encephalitis virus RNA from a cDNA clone: analysis of a viable deletion mutant. Virology. 1989 Jul; 171(1); 189-204. [PubMed: 2525837].

10. Davis et al., 1991: Davis NL, Powell N, Greenwald GF, Willis LV, Johnson BJ, Smith JF, Johnston RE. Attenuating mutations in the E2 glycoprotein gene of Venezuelan equine encephalitis virus: construction of single and multiple mutants in a full-length cDNA clone. Virology. 1991 Jul; 183(1); 20-31. [PubMed: 2053280 ].

11. Davis et al., 1995: Davis NL, Brown KW, Greenwald GF, Zajac AJ, Zacny VL, Smith JF, Johnston RE. Attenuated mutants of Venezuelan equine encephalitis virus containing lethal mutations in the PE2 cleavage signal combined with a second-site suppressor mutation in E1. Virology. 1995; 212(1); 102-110. [PubMed: 7676619].

12. Davis et al., 1996: Davis NL, Brown KW, Johnston RE. A viral vaccine vector that expresses foreign genes in lymph nodes and protects against mucosal challenge. Journal of virology. 1996 Jun; 70(6); 3781-7. [PubMed: 8648713].

13. Dupuy et al., 2009: Dupuy LC, Locher CP, Paidhungat M, Richards MJ, Lind CM, Bakken R, Parker MD, Whalen RG, Schmaljohn CS. Directed molecular evolution improves the immunogenicity and protective efficacy of a Venezuelan equine encephalitis virus DNA vaccine. Vaccine. 2009; 27(31); 4152-4160. [PubMed: 19406186].

14. Eddy et al., 1972: Eddy GA, Martin DH, Reeves WC, Johnson KM. Field studies of an attenuated Venezuelan equine encephalomyelitis vaccine (strain TC-83). Infection and immunity. 1972 Feb; 5(2); 160-3. [PubMed: 4564397].

15. Fine et al., 2007: Fine DL, Roberts BA, Teehee ML, Terpening SJ, Kelly CL, Raetz JL, Baker DC, Powers AM, Bowen RA. Venezuelan equine encephalitis virus vaccine candidate (V3526) safety, immunogenicity and efficacy in horses. Vaccine. 2007 Feb 26; 25(10); 1868-76. [PubMed: 17240002].

16. Grieder et al., 1995: Grieder FB, Davis NL, Aronson JF, Charles PC, Sellon DC, Suzuki K, Johnston RE. Specific restrictions in the progression of Venezuelan equine encephalitis virus-induced disease resulting from single amino acid changes in the glycoproteins. Virology. 1995 Feb 1; 206(2); 994-1006. [PubMed: 7856110].

17. Guyton, A.C., 1947: Guyton, A.C.. Measurement of the respiratory volume of laboratory animals. Am. J. Physiol.. 1947; 150; 10-11.

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