VIOLIN: Vaccine Investigation and Online Information Network

Variola virus

Table of Contents

General Information
Vaccine Related Pathogen Genes
1. B14R
2. A27L
3. A33R from Monkeypox virus (strain: Zaire-96-I-16)
4. A33R from Vaccinia virus (strain: WR (Western Reserve))
5. A34R
6. A36R
7. A56R
8. B5R from Monkeypox virus (strain: Zaire-96-I-16)
9. B5R from Vaccinia virus (strain: WR (Western Reserve))
10. D8L
11. F13L
12. L1R from Vaccinia virus (strain: WR (Western Reserve))
13. p53
14. VACVgp196
15. VACVgp200
16. A21L (Protective antigen)
17. A30L (Protective antigen)
18. H3L (Protective antigen)
19. L1R from Monkeypox virus Zaire-96-I-16 (Protective antigen)
Vaccine Information
References

I. General Information

1. NCBI Taxonomy ID:

10255

2. Disease:

Smallpox

3. Introduction

The poxviruses are a family of large, enveloped deoxyribonucleic acid (DNA) viruses. The most notorious poxvirus is variola, the causative agent of smallpox. Smallpox was an important cause of morbidity and mortality in the developing world until recent times. Since the host range of the variola virus is confined to humans, aggressive case identification and contact vaccination were ultimately successful in controlling the disease. The last occurrence of endemic smallpox was in Somalia in 1977, and the last human cases were laboratory-acquired infections in 1978. By 1980, the World Health Organization (WHO) General Assembly ratified the declaration of success made by the Global Commission for the Certification of Smallpox Eradication (PathPort).

4. Microbial Pathogenesis

Smallpox is a viral disease unique to humans. To sustain itself, the virus must pass from person to person in a continuing chain of infection and is spread by inhalation of air droplets or aerosols. There are three principal routes of viral infection corresponding to the three principal surfaces of the body: the respiratory tract, the alimentary tract, and the skin. Minor routes of infection include the urinary and genital tracts and the conjunctiva. Although congenital infection occasionally occurred in smallpox, it was of no epidemiologic importance. The infectious dose is unknown but is believed to be only a few virions (Henderson, 1999).
It is assumed to be low, 10 to 100 organisms (Franz et al., 1997).
Variola is most effectively spread via the respiratory route with as little as ten plaque-forming units contained within aerosolized saliva able to transmit the infection from person to person (Hassett, 2003).
Variola virus is highly stable and retains its infectivity for long periods outside the host. It is infectious by aerosol, but natural airborne spread to other than close contacts is controversial. Approximately 30% of susceptible contacts became infected during the era of endemic smallpox, and the WHO eradication campaign was predicated on close person-to-person proximity being required for transmission to occur reliably. Nevertheless, variola virus's potential in low relative humidity for airborne dissemination was alarming in two hospital outbreaks. On natural exposure to aerosolized virus, variola travels from the upper or the lower respiratory tract to regional lymph nodes, where it replicates and gives rise to viremia, which is followed soon thereafter by a rash (PathPort).

5. Host Ranges and Animal Models

Variola virus is considered to be a host-restricted poxvirus, with humans as the reservoir host and no zoonotic hosts known. However, the virus does replicate well in most mammalian cell cultures (McFadden, 2005).

6. Host Protective Immunity

Both cellular and humoral immune response are important in protection against smallpox. In mouse models of vaccinia infection, extensive studies have shown that passive immunotherapy with immune serum or monoclonal antibodies are protective. Antiviral antibody could protect mice efficiently even if CD4+ or CD8+ T cells were depleted prior to challenge. However, in B-cell-deficient or MHC II-deficient mice, which are unable to elicit effective antibody responses, strong antiviral T-cell responses played an important role in protecting against disease following viral challenge. Studies using non-human primates infected with lethal monkeypox showed that the smallpox vaccine-induced antibody responses were both necessary and sufficient for protection against lethal monkeypox infection. In humans, cellular immunity played the most important role in protective immunity in humans against primary poxvirus infections based on the severe complications following smallpox vaccination of children with genetic T-cell deficiencies. Following vaccination, strong antiviral antibody responses are likely to be the main effector mechanism responsible for protection against secondary infection (Amanna et al., 2006).

II. Vaccine Related Pathogen Genes

1. A21L

Gene Name : A21L
Sequence Strain (Species/Organism) : Vaccinia virus
VO ID : VO_0012363
NCBI Gene ID : 3707670
NCBI Protein GI : 66275937
Locus Tag : VACWR140
Genbank Accession : AY603355
Protein Accession : YP_233022
Taxonomy ID : 10245
Gene Starting Position : 127904
Gene Ending Position : 128257
Gene Strand (Orientation) : -
Protein Name : IMV membrane protein
Protein pI : 8.71
Protein Weight : 12850.65
Protein Length : 117
Protein Note : IMV membrane protein; similar to vaccinia virus strain Copenhagen A21L; The poxviridae are enveloped unsegmented dsDNA viruses; unlike many dsDNA viruses that replicate in the host nucleus poxviruses encode their own replication machinery and therefore replicate in the cytoplasm; viral genes are expressed in a bi-phasic manner with early genes encoding non-structural proteins involved in genome replication and late genes encoding the viral structural proteins

DNA Sequence : Show Sequence

>NC_006998.1:127904-128257 Vaccinia virus, complete genome
TTTAGGTAGTAAAAAATAAGTCAGAATATGCCCTATAACACGATCGTGCAAAACCTGGTATATCGTCTCT
ATCTTTATCACAATATAGTGTATCGACATCTTTATTATTATTGACCTCGTTTATCTTGGAACATGGAATG
GGAACATTTTTGTTATCAACGGCCACCTTTGCCTTAATTCCAGATGTTGTAAAATTATAACTAAACAGTC
TATCATCGACACAAATGAAATTCTTGTTTAGACGTTTGTAGTTTACGTATGCGGCTCGTTCGCGTCTCAT
TTTTTCAGATATTGCAGGTACTATAATATTAAAAATAAGAATGAAATAACATAGGATTAAAAATAAAGTT
ATCA

Protein Sequence : Show Sequence

>YP_233022.1 Membrane protein [Vaccinia virus]
MITLFLILCYFILIFNIIVPAISEKMRRERAAYVNYKRLNKNFICVDDRLFSYNFTTSGIKAKVAVDNKN
VPIPCSKINEVNNNKDVDTLYCDKDRDDIPGFARSCYRAYSDLFFTT

Molecule Role : Protective antigen
Related Vaccine(s): LC16m8

2. A27L

Gene Name : A27L
Sequence Strain (Species/Organism) : Monkeypox virus (strain: Zaire-96-I-16)
NCBI Gene ID : 928973
Locus Tag : MPXVgp137
Gene Strand (Orientation) : ?

DNA Sequence : Show Sequence

>gi|17529780:c137905-135815 Monkeypox virus strain Zaire-96-I-16, complete genome
ATGGAGGTCACGAACCTTATTAAAAAATGTACCAAACACTCCAAAGATTTCGCCACTGAGGTAGAAAAAC
TATGGAATGATGAGTTGAGTTCTGAATCAGGTCTCACAAGAAAAACAAGAAATGTAATTCGTAATATTCT
TCGTGATATCACTAAGTCATTAACTACAGATAAGAAATCAAAGTGTTTCCGTATACTAGAACGTTCGACG
ATTAACGGAGAGCAGATTAAAGATGTATATAAAACTATTTTTAATAATGGTGTTGATGTGGAGTCTAGAA
TCAACACTACAGGAAAGTATGTTCTATTTACAGTTATGACTTATGCTGCTGAACTACATCTCATTAAGTC
AGACGAGATATTCGCTCTTCTATCAAGATTTTTTAACATGATATGTGATATTCATAGAAAATACGGATGT
GGTAATATGTTTGTTGGTATTCCTGCCGCTCTAATTGTTCTGTTGGAAATTGATCACATCAATAAACTGT
TTAGCGTGTTTAGTACAAGATATGATGCTAAGGCATATCTATATACTGAATATTTCCTCTTCCTTAACAT
TAATCATTATCTACTTAGTGGTTCAGATCTATTTATCAACGTAGCATATGGTGCTGTATCTTTTTCGTCA
CCCATTAGTGTTCCAGACTATATCATGGAAGCATTGACATTTAAGGCATGCGATCATATTATGAAATCTG
GAGATCTAAAATATACATATGCATTTACTAAAAAGGTCAAGGATCTGTTTAATACTAAATCTGATTCTGT
TTATCAATACGTTAGACTTCATGAAATGTCATATGATGGCGTTTCAGAAGATACGGATGATGACGATGAG
GTATTCGCTATCCTTAACTTGAGTATCGATTCCAGCGTTGATAGATACAGAAACAGAGTTCTTCTACTAA
CTCCTGAAGTCGCGTCTCTTAGAAAAGAATATTCTGAAACAGAACCCGATTATAAATACTTGATGGATGA
GGAAGTGCCCGCGTACGACAAGCATTTGTCTAAGCCTATTACTAATACTGGTATTGAAGAACCACATGCT
ACTGGAGGAGATAAGGAGGACCAACCAATTAAGGTTGTCCATCCCCCACCTAATAATGATAAAGATGATG
CTATCAAGCCATACAATCCATTAGAAGATCCTAATTATGTTCCCACAAATACAAGAACGGTTATAGGAAT
CGCTGATTACCAACTAGTCATTAATAAACTAATTGAATGGTTAGATAAATGCGAGGAAGAATGCGGAAAT
GATGGAGAGTTTAAAACAGACTTGGAAGAAGCCAAGAGAAAACTCACTGAATTGAATGAAGAACTTAGTG
ATAAACTCAGTAAGATTAGGACTTTGGAAAGGGATTCTGTTTATAAAACCGAAAGAATCGACAGACTTAC
AACAGAGATCAAAGAACTCAGGGATATGCAACAAAATGGGACAGATGATGGTTCAGATTCATCAGAAATT
GATAAGAAGACTATACGAGAATTGAGAGAATCTCTTGATATGGAACGAGAAATGCGGTCAGAACTAGAAA
AGGAACTGGATACTATTAGGGATGGAAAAGTAGATGGATCTTGTCAACGAGAACTTGAACTCAGTCGTAT
GTGGCTAAAACAACGCGACGACGATCTCCGAGCTGAAATCGATAAACGTCGTAATGTCGAATGGGAACTG
TCCAGACTTCGTATGGATATCAAGGAATGTGACAAATACAAGGAGGATCTTGATAAGGATAAGACAACTA
TTAGTACATACGTGAGCAGAATCAGTACTCTAGAATCAGAAATTGCTAAATATCAACAAGATAGGGACAC
GCTTTCTGTAGTACACAGAGAACTTGAGGAAGAACGACGACACGTTAGAGATCTCGAATCTAGACTCGAT
GAATGCACACGCAATCAAGAAGACACACAAGAAGTTGATGCACTGCGTTCACGTATTAGAGAACTAGAGA
ATAAGTTGACCGACTGCATCGAGAGCGGAGGAGGAAATCTTACAGAGATTAGCAGACTCCAATCTAGAAT
CTCAGATCTTGAAAGACAACTGAGTGAATGCCGTGGAAATGCTACAGAGATTACAATCTAG

Protein Sequence : Show Sequence

>gi|17529917:1-696 A27L [Monkeypox virus]
MEVTNLIKKCTKHSKDFATEVEKLWNDELSSESGLTRKTRNVIRNILRDITKSLTTDKKSKCFRILERST
INGEQIKDVYKTIFNNGVDVESRINTTGKYVLFTVMTYAAELHLIKSDEIFALLSRFFNMICDIHRKYGC
GNMFVGIPAALIVLLEIDHINKLFSVFSTRYDAKAYLYTEYFLFLNINHYLLSGSDLFINVAYGAVSFSS
PISVPDYIMEALTFKACDHIMKSGDLKYTYAFTKKVKDLFNTKSDSVYQYVRLHEMSYDGVSEDTDDDDE
VFAILNLSIDSSVDRYRNRVLLLTPEVASLRKEYSETEPDYKYLMDEEVPAYDKHLSKPITNTGIEEPHA
TGGDKEDQPIKVVHPPPNNDKDDAIKPYNPLEDPNYVPTNTRTVIGIADYQLVINKLIEWLDKCEEECGN
DGEFKTDLEEAKRKLTELNEELSDKLSKIRTLERDSVYKTERIDRLTTEIKELRDMQQNGTDDGSDSSEI
DKKTIRELRESLDMEREMRSELEKELDTIRDGKVDGSCQRELELSRMWLKQRDDDLRAEIDKRRNVEWEL
SRLRMDIKECDKYKEDLDKDKTTISTYVSRISTLESEIAKYQQDRDTLSVVHRELEEERRHVRDLESRLD
ECTRNQEDTQEVDALRSRIRELENKLTDCIESGGGNLTEISRLQSRISDLERQLSECRGNATEITI

Related Vaccine(s): Smallpox DNA Vaccine

3. A30L

Gene Name : A30L
Sequence Strain (Species/Organism) : Variola virus
NCBI Protein GI : CAA47504
Other Database IDs : CDD:283633
InterPro: IPR009257
UniProtKB/UniProt: P68597
Taxonomy ID : 10255
Protein Name : A30L
Protein pI : 9.27
Protein Weight : 8777.13
Protein Length : 113
Protein Note : Chordopoxvirus A30L protein; pfam06015

Protein Sequence : Show Sequence

>CAA47504.1 A30L [Variola virus]
MEDLNEANFSHLLINLSNNKDIDAQYASTLSVVHELLSAINFKIFNINKKSKKNSKSIEQHPVVHHAASA
GREFNRR

Molecule Role : Protective antigen
Molecule Role Annotation : (Maksiutov and Shchelkunov, 2011)

4. A33R from Monkeypox virus (strain: Zaire-96-I-16)

Gene Name : A33R from Monkeypox virus (strain: Zaire-96-I-16)
Sequence Strain (Species/Organism) : Monkeypox virus (strain: Zaire-96-I-16)
NCBI Gene ID : 928961
Locus Tag : MPXVgp143

DNA Sequence : Show Sequence

>gi|17529780:141611-142039 Monkeypox virus strain Zaire-96-I-16, complete genome
ATGGCATCTATTTTAAATACACTTAGGTTTTTAGAAAAAACATCATTTTATAATTGTAACGATTCAATAA
CTAAAGAAAAGATTAAGATTAAACATAAGGGAATGTCATTTGTATTTTATAAGCCAAAGCATTCTACCGT
TGTTAAATACTTGTCTGGAGGAGGTATATATCATGATGATTTGGTTGTATTGGGGAAGGTAACAATTAAT
GATCTAAAGATGATGCTATTTTACATGGATTTATCATATCATGGAGTGACAAGTAGTGGAGCAATTTACA
AATTGGGATCGTCTATCGATAGACTTTCTCTAAATAGGACTATTGTTACAAAAGTTAATAACAATTATAA
CAATTATAACAATTATAATTGTTATAATAATTATAATTGTTATAATTATGATGATACATTTTTTGACGAT
GATGATTGA

Protein Sequence : Show Sequence

>gi|17529923:1-142 A33R [Monkeypox virus]
MASILNTLRFLEKTSFYNCNDSITKEKIKIKHKGMSFVFYKPKHSTVVKYLSGGGIYHDDLVVLGKVTIN
DLKMMLFYMDLSYHGVTSSGAIYKLGSSIDRLSLNRTIVTKVNNNYNNYNNYNCYNNYNCYNYDDTFFDD
DD

Related Vaccine(s): Smallpox DNA Vaccine

5. A33R from Vaccinia virus (strain: WR (Western Reserve))

Gene Name : A33R from Vaccinia virus (strain: WR (Western Reserve))
Sequence Strain (Species/Organism) : Vaccinia virus (strain: WR (Western Reserve))
NCBI Gene ID : 3707686
Locus Tag : VACWR156
Genbank Accession : NC_006998
Gene Strand (Orientation) : ?

DNA Sequence : Show Sequence

>gi|66275797:143331-143888 Vaccinia virus, complete genome
ATGATGACACCAGAAAACGACGAAGAGCAGACATCTGTGTTCTCCGCTACTGTTTACGGAGACAAAATTC
AAGGAAAGAATAAACGCAAACGCGTGATTGGTCTATGTATTAGAATATCTATGGTTATTTCACTACTATC
TATGATTACCATGTCCGCGTTTCTCATAGTGCGCCTAAATCAATGCATGTCTGCTAACGAGGCTGCTATT
ACTGACGCCGCTGTTGCCGTTGCTGCTGCATCATCTACTCATAGAAAGGTTGCGTCTAGCACTACACAAT
ATGATCACAAAGAAAGCTGTAATGGTTTATATTACCAGGGTTCTTGTTATATATTACATTCAGACTACCA
GTTATTCTCGGATGCTAAAGCAAATTGCACTGCGGAATCATCAACACTACCCAATAAATCCGATGTCTTG
ATTACCTGGCTCATTGATTATGTTGAGGATACATGGGGATCTGATGGTAATCCAATTACAAAAACTACAT
CCGATTATCAAGATTCTGATGTATCACAAGAAGTTAGAAAGTATTTTTGTGTTAAAACAATGAACTAA

Protein Sequence : Show Sequence

>gi|222720|dbj|BAA01805.1| 20.5K protein [Vaccinia virus]
MMTPENDEEQTSVFSATVYGDKIQGKNKRKRVIGLCIRISMVISLLSMITMSAFLIVRLNQCMSANEAAI
TDAAVAVAAASSTHRKVASSTTQYDHKESCNGLYYQGSCYILHSDYQLFSDAKANCTAESSTLPNKSDVL
ITWLIDYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCVKTMN

Related Vaccine(s): IMV-EEV , LC16m8

6. A34R

Gene Name : A34R
Sequence Strain (Species/Organism) : Vaccinia virus (strain: WR (Western Reserve))
NCBI Gene ID : 3707687
Locus Tag : VACWR157
Genbank Accession : NC_006998
Gene Strand (Orientation) : ?
Protein Note : EEV glycoprotein

DNA Sequence : Show Sequence

>gi|66275797:143912-144418 Vaccinia virus, complete genome
ATGAAATCGCTTAATAGACAAACTGTAAGTAGGTTTAAGAAGTTGTCGGTGCCGGCCGCTATAATGATGA
TACTCTCAACCATTATTAGTGGCATAGGAACATTTCTGCATTACAAAGAAGAACTGATGCCTAGTGCTTG
CGCCAATGGATGGATACAATACGATAAACATTGTTATTTAGATACTAACATTAAAATGTCTACAGATAAT
GCGGTTTATCAGTGTCGTAAATTACGAGCCAGATTGCCTAGACCGGATACTAGACATCTGAGAGTATTGT
TTAGTATTTTTTATAAAGATTATTGGGTAAGTTTAAAAAAGACCAATGATAAATGGTTAGATATTAATAA
TGATAAAGATATAGATATTAGTAAATTAACAAATTTTAAACAACTAAACAGTACGACGGATGCTGAAGCG
TGTTATATATACAAGTCTGGAAAACTGGTTAAAACAGTATGTAAAAGTACTCAATCTGTACTATGTGTTA
AAAAATTCTACAAGTGA

Protein Sequence : Show Sequence

>gi|222721|dbj|BAA01806.1| 19.5K protein [Vaccinia virus]
MKSLNRQTVSRFKKLSVPAAIMMILSTIISGIGTFLHYKEELMPSACANGWIQYDKHCYLDTNIKMSTDN
AVYQCRKLRARLPRPDTRHLRVLFSIFYKDYWVSLKKTNDKWLDINNDKDIDISKLTNFKQLNSTTDAEA
CYIYKSGKLVKTVCKSTQSVLCVKKFYK

Related Vaccine(s): IMV-EEV , LC16m8

7. A36R

Gene Name : A36R
Sequence Strain (Species/Organism) : Variola virus
NCBI Gene ID : 3707689
Locus Tag : VACWR159
Gene Strand (Orientation) : ?
Protein Note : IEV transmembrane phosphoprotein

DNA Sequence : Show Sequence

>gi|66275797:145059-145724 Vaccinia virus, complete genome
ATGATGCTGGTACCTCTTATCACGGTGACCGTAGTTGCGGGAACAATATTAGTATGTTATATATTATATA
TTTGTAGGAAAAAGATACGTACTGTCTATAATGACAATAAAATTATCATGACAAAATTAAAAAAGATAAA
GAGTTCTAATTCCAGCAAATCTAGTAAATCAACTGATAGCGAATCAGACTGGGAGGATCACTGTAGTGCT
ATGGAACAAAACAATGACGTAGATAATATTTCTAGGAATGAGATATTGGACGATGATAGCTTCGCTGGTA
GTTTAATATGGGATAACGAATCCAATGTCATGGCGCCTAGCACAGAACACATTTACGATAGTGTTGCTGG
AAGCACGCTGCTAATAAATAATGATCGTAATGAACAGACTATTTATCAGAACACTACAGTAGTAATTAAT
GAGACGGAGACTGTTGAAGTACTTAATGAAGATACCAAACAGAATCCTAACTATTCATCCAATCCTTTCG
TAAATTATAATAAAACCAGTATTTGTAGCAAGTCAAATCCGTTCATTACAGAACTCAACAATAAATTTAG
TGAGAATAATCCGTTTAGACGAGCACATAGCGATGATTATCTTAATAAGCAAGAACAAGATCATGAACAC
GATGATATAGAATCATCGGTCGTATCATTGGTGTGA

Protein Sequence : Show Sequence

>gi|222723|dbj|BAA01808.1| 25.1K protein [Vaccinia virus]
MMLVPLITVTVVAGTILVCYILYICRKKIRTVYNDNKIIMTKLKKIKSSNSSKSSKSTDSESDWEDHCSA
MEQNNDVDNISRNEILDDDSFAGSLIWDNESNVMAPSTEHIYDSVAGSTLLINNDRNEQTIYQNTTVVIN
ETETVEVLNEDTKQNPNYSSNPFVNYNKTSICSKSNPFITELNNKFSENNPFRRAHSDDYLNKQEQDHEH
DDIESSVVSLV

Related Vaccine(s): IMV-EEV , LC16m8

8. A56R

Gene Name : A56R
Sequence Strain (Species/Organism) : Vaccinia virus (strain: WR (Western Reserve))
NCBI Gene ID : 3707652
Locus Tag : VACWR181

DNA Sequence : Show Sequence

>gi|335637|gb|M93956.1|VACHGG Vaccinia virus hemagglutinin gene, complete cds
TTGGACATTGGATAATGGTCACGTGTTACCACGCAATTATATAATGTATAAATGCGAACCGATTAAACAT
AAATATCCATTGGAAAAAACACAGTACACGAATGATTTTCTAAAGTATTTGGAAAGTTTTATAGGTAGTT
GATAGAACAAAATACATAATTTTGTAAAAATAAATCACTTTTTATACTAATATGACACGATTACCAATAC
TTTTGTTACTAATATCATTAGTATACGCTACACCTTTTCCTCAGACATCTAAAAAAATAGGTGATGATGC
AACTCTATCATGTAATCGAAATAATACAAATGACTACGTTGTTATGAGTGCTTGGTATAAGGAGCCCAAT
TCCATTATTCTTTTAGCTGCTAAAAGCGACGTCTTGTATTTTGATAATTATACCAAGGATAAAATATCTT
ACGACTCTCCATACGATGATCTAGTTACAACTATCACAATTAAATCATTGACTGCTAGAGATGCCGGTAC
TTATGTATGTGCATTCTTTATGACATCAACTACAAATGACACTGATAAAGTAGATTATGAAGAATACTCC
ACAGAGTTGATTGTAAATACAGATAGTGAATCGACTATAGACATAATACTATCTGGATCTACACATTCAC
CGGAAACTAGTTCTAAGAAACCTGATTATATAGATAATTCTAATTGCTCGTCGGTATTCGAAATCGCGAC
TCCGGAACCAATTACTGATAATGTAGAAGATCATACAGACACCGTCACATACACTAGTGATAGCATTAAT
ACAGTAAGTGCATCATCTGGAGAATCCACAACAGACGAGACTCCGGAACCAATTACTGATAAAGAAGATC
ATACAGTTACAGACACTGTCTCATACACTACAGTAAGTACATCATCTGGAATTGTCACTACTAAATCAAC
CACCGATGATGCGGATCTTTATGATACGTACAATGATAATGATACAGTACCACCAACTACTGTAGGCGGT
AGTACAACCTCTATTAGCAATTATAAAACCAAGGACTTTGTAGAAATATTTGGTATTACCGCATTAATTA
TATTGTCGGCCGTGGCAATATTCTGTATTACATATTATATATATAATAAACGTTCACGTAAATACAAAAC
AGAGAACAAAGTCTAGATTTTTGACTTACATAAATGTCTGGGATAGTAAAATCTATCATATTGAGCGGAC
CATCTGGTTTAGGAAAGACAGCCATAGCCAAAAGACTATGGGAATATATTTGGATTTGTGGTGTCCCATA
CCACTAGATTTCCTCGTCCTATGGAACGAGAAGGTGTTGATTACCATTACGTTAACAGAGAGGCCATCTG
GAAGGGAATAGCCGCCGGAAACTTTCTAGAACATACTGAGTTTTTAGGAAATATTTACGGAACTTCTAAA
ACAGCTGTGAATACACGGCTATTAATAATCGTATTTGTGTGATGGATCTAAACATCGACGGTGTTAGAAG
TCTTAAAAATACGTACCTAATGCCTTACTCGGTGTATATAAGACCTACCTCTCTTAAAATGGTTGAGACC
AAGCTTGGCACTGGCGTCGTTT

Protein Sequence : Show Sequence

>gi|335638|gb|AAA48252.1| hemagglutinin
MTRLPILLLLISLVYATPFPQTSKKIGDDATLSCNRNNTNDYVVMSAWYKEPNSIILLAAKSDVLYFDNY
TKDKISYDSPYDDLVTTITIKSLTARDAGTYVCAFFMTSTTNDTDKVDYEEYSTELIVNTDSESTIDIIL
SGSTHSPETSSKKPDYIDNSNCSSVFEIATPEPITDNVEDHTDTVTYTSDSINTVSASSGESTTDETPEP
ITDKEDHTVTDTVSYTTVSTSSGIVTTKSTTDDADLYDTYNDNDTVPPTTVGGSTTSISNYKTKDFVEIF
GITALIILSAVAIFCITYYIYNKRSRKYKTENKV

Related Vaccine(s): LC16m8

9. B14R

Gene Name : B14R
NCBI Gene ID : 8384129

10. B5R from Monkeypox virus (strain: Zaire-96-I-16)

Gene Name : B5R from Monkeypox virus (strain: Zaire-96-I-16)
Sequence Strain (Species/Organism) : Monkeypox virus (strain: Zaire-96-I-16)
NCBI Gene ID : 928916
Locus Tag : MPXVgp166

DNA Sequence : Show Sequence

>gi|17974913:163058-164743 Monkeypox virus, complete genome
ATGGATTTTTTTAAAAAGGAAATACTTGACTGGAGTATATATTTATTTCTTCATTACATAACACGTCTGT
GTTCTAATTCTTCCAATTCTTCCACATCTCATATAATACAGGAATATAATCTTGTTCGAAAATACGAGAA
AGTGGATAAAACAATAGTTGATTTTTTATCTAGGTGGCCAAATTTATTCCATATTTTAGAATATGGGGAA
AATATTCTACATATTTATTTTATAGATGCTGCTAATACGAATATTATGATTTTTTTTCTAGATAGAGTAT
TAAATATTAATAAGAACCGTGGGTCATTTATACATAATCTCGGGTTATCATCCATTAATATAAAAGAATA
TGTATATCAATTAGTTAATAATGATCATCTAGATAATAGTATAAGACTAATGCTTGAAAATGGACGTAGA
ACAAGACATTTTTTGTCTTATATATTGGATACAGTTAATATCTATATAAGTATTTTAATAAATCATAGAT
TTTATATAGATGCCGAAGACAGTTACGGTTGTACATTATTACATAGATGTATATATAACTATAGGAAATC
AGAATCAGAATCATATAATGAATTAATTAAGATATTGTTAAATAATGGATCAGATGTAGATAAAAAAGAT
ACGTACGGAAACACACCGTTTATCCTATTATGTAAACACGATATCGACAACGCGGAATTGTTTGAGATAT
GTTTAGAGAATGCTAATATAGACTCTGTAGACTTTAATGGATATACACCTCTTCATTATGTCTCATGTCG
TAATAAATATGATTTTGTAAAGTTATTAATTTCTAAAGGAGCAAATGTTAATGCACGTAATAGATTCGGA
ACTACTCCATTTTATTGTGGAATTATACACGGTATCTCGCTTATAAAACTATATTTGGAATCAGACACAG
AGTTAGAAATAGATAATGAACATATAGTTCGTCATTTAATAATTTTTGATGCTGTTGAATCTTTAGATTA
TCTATTGTCCAGAGGAGTTATTGATATTAACTATCGTACTATATACAACGAAACATCTATTTACGACGCT
GTCAGTTATAATGCGTATAATACGTTAGTCTATCTATTAAACAGAAATGGTGATTTTGAGACGATTACTA
CTAGTGGATGTACATGTATTTCGGAAGCAGTCGCGAACAACAACAAAATAATAATGGATATACTATTGTC
TAAACGACCATCTTTGAAAATTATGATACCATCTATGATAGCAATTACTAAACATAAACAACATAATGCA
GATTTATTGAAAATGTGTATAAAATATACTGCGTGTATGACCGATTATGATACTCTTATAGATGTACAAT
CGCTACATCAATATAAATGGTATATTTTAAAATGTTTTGATGAAATAGATATCATGAAGAGATGTTATAT
AAAAAATAAAACTGTATTCCAATTAGTTTTTTGTATCAAAGACATTAATACTTTAATGAGATACGGTAGA
CATCCTTCTTTCGTGAAATGTAATATTCTCGACGTATACGGAAGTCATGTACGTAATATCATAGCATCTA
TTAGATATCGTCAGAGATTAATTAGTCTATTATCCAAGAAGCTGGATGCTGGAGATAAATGGTCGTGTTT
TCCTAACGAAATAAAATATAAAATATTGGAAAACTTTAACGATAACGAACTGACCACATATCTAAAAATC
TTATAA

Related Vaccine(s): Smallpox DNA Vaccine

11. B5R from Vaccinia virus (strain: WR (Western Reserve))

Gene Name : B5R from Vaccinia virus (strain: WR (Western Reserve))
Sequence Strain (Species/Organism) : Vaccinia virus (strain: WR (Western Reserve))
NCBI Gene ID : 3707658
Locus Tag : VACWR187
Gene Strand (Orientation) : ?
Protein Note : EEV type-I membrane glycoprotein

DNA Sequence : Show Sequence

>gi|66275797:168374-169327 Vaccinia virus, complete genome
ATGAAAACGATTTCCGTTGTTACGTTGTTATGCGTACTACCTGCTGTTGTTTATTCAACATGTACTGTAC
CCACTATGAATAACGCTAAATTAACGTCTACCGAAACATCGTTTAATGATAAACAGAAAGTTACGTTTAC
ATGTGATCAGGGATATCATTCTTCGGATCCAAATGCTGTCTGCGAAACAGATAAATGGAAATACGAAAAT
CCATGCAAAAAAATGTGCACAGTTTCTGATTACATCTCTGAATTATATAATAAACCGCTATACGAAGTGA
ATTCCACCATGACACTAAGTTGCAACGGCGAAACAAAATATTTTCGTTGCGAAGAAAAAAATGGAAATAC
TTCTTGGAATGATACTGTTACGTGTCCTAATGCGGAATGTCAACCTCTTCAATTAGAACACGGATCGTGT
CAACCAGTTAAAGAAAAATACTCATTTGGGGAATATATGACTATCAACTGTGATGTTGGATATGAGGTTA
TTGGTGCTTCGTACATAAGTTGTACAGCTAATTCTTGGAATGTTATTCCATCATGTCAACAAAAATGTGA
TATGCCGTCTCTATCTAATGGATTAATTTCCGGATCTACATTTTCTATCGGTGGCGTTATACATCTTAGT
TGTAAAAGTGGTTTTACACTAACGGGGTCTCCATCATCCACATGTATCGACGGTAAATGGAATCCCGTAC
TCCCAATATGTGTACGAACTAACGAAGAATTTGATCCAGTGGATGATGGTCCCGACGATGAGACAGATTT
GAGCAAACTCTCGAAAGACGTTGTACAATATGAACAAGAAATAGAATCGTTAGAAGCAACTTATCATATA
ATCATAGTGGCGTTAACAATTATGGGCGTCATATTTTTAATCTCCGTTATAGTATTAGTTTGTTCCTGTG
ACAAAAATAATGACCAATATAAGTTCCATAAATTGCTACCGTAA

Protein Sequence : Show Sequence

>gi|222750|dbj|BAA01835.1| 35.1K protein [Vaccinia virus]
MKTISVVTLLCVLPAVVYSTCTVPTMNNAKLTSTETSFNDKQKVTFTCDQGYHSSDPNAVCETDKWKYEN
PCKKMCTVSDYISELYNKPLYEVNSTMTLSCNGETKYFRCEEKNGNTSWNDTVTCPNAECQPLQLEHGSC
QPVKEKYSFGEYMTINCDVGYEVIGASYISCTANSWNVIPSCQQKCDMPSLSNGLISGSTFSIGGVIHLS
CKSGFTLTGSPSSTCIDGKWNPVLPICVRTNEEFDPVDDGPDDETDLSKLSKDVVQYEQEIESLEATYHI
IIVALTIMGVIFLISVIVLVCSCDKNNDQYKFHKLLP

Related Vaccine(s): IMV-EEV , LC16m8

12. D8L

Gene Name : D8L
Sequence Strain (Species/Organism) : Vaccinia virus (strain: WR (Western Reserve))
NCBI Gene ID : 3707569
Locus Tag : VACWR113
Gene Strand (Orientation) : ?
Protein Note : IMV membrane protein

DNA Sequence : Show Sequence

>gi|66275797:c103975-103061 Vaccinia virus, complete genome
ATGCCGCAACAACTATCTCCTATTAATATAGAAACTAAAAAAGCAATTTCTAACGCGCGATTGAAGCCGT
TAGACATACATTATAATGAGTCGAAACCAACCACTATCCAGAACACTGGAAAACTAGTAAGGATTAATTT
TAAAGGAGGATATATAAGTGGAGGGTTTCTCCCCAATGAATATGTGTTATCATCACTACATATATATTGG
GGAAAGGAAGACGATTATGGATCCAATCACTTGATAGATGTGTACAAATACTCTGGAGAGATTAATCTTG
TTCATTGGAATAAGAAAAAATATAGTTCTTATGAAGAGGCAAAAAAACACGATGATGGACTTATCATTAT
TTCTATATTCTTACAAGTATTGGATCATAAAAATGTATATTTTCAAAAGATAGTTAATCAATTGGATTCC
ATTAGATCCGCCAATACGTCTGCACCGTTTGATTCAGTATTTTATCTAGACAATTTGCTGCCTAGTAAGT
TGGATTATTTTACATATCTAGGAACAACTATCAACCACTCTGCAGACGCTGTATGGATAATTTTTCCAAC
GCCAATAAACATTCATTCTGATCAACTATCTAAATTCAGAACACTATTGTCGTCGTCTAATCATGATGGA
AAACCGCATTATATAACAGAGAACTATAGAAATCCGTATAAATTGAACGACGACACGCAAGTATATTATT
CTGGGGAGATTATACGAGCAGCAACTACCTCTCCAGCGCGCGAGAACTATTTTATGAGATGGTTGTCCGA
TTTGAGAGAGACATGTTTTTCATATTATCAAAAATATATCGAAGAGAATAAAACATTCGCAATTATTGCC
ATAGTATTCGTGTTTATACTTACCGCTATTCTCTTTTTTATGAGTCGACGATATTCGCGAGAAAAACAAA
ACTAG

Protein Sequence : Show Sequence

>gi|335652|gb|AAA48264.1| ORF8 cds
MPQQLSPINIETKKAISNARLKPLDIHYNESKPTTIQNTGKLVRINFKGGYISGGFLPNEYVLSSLHIYW
GKEDDYGSNHLIDVYKYSGEINLVHWNKKKYSSYEEAKKHDDGLIIISIFLQVLDHKNVYFQKIVNQLDS
IRSANTSAPFDSVFYLDNLLPSKLDYFTYLGTTINHSADAVWIIFPTPINIHSDQLSKFRTLLSSSNHDG
KPHYITENYRNPYKLNDDTQVYYSGEIIRAATTSPARENYFMRWLSDLRETCFSYYQKYIEENKTFAIIA
IVFVFILTAILFFMSRRYSREKQN

Related Vaccine(s): IMV-EEV

13. F13L

Gene Name : F13L
Sequence Strain (Species/Organism) : Vaccinia virus (strain: WR (Western Reserve))
NCBI Gene ID : 3707509
Locus Tag : VACWR052

DNA Sequence : Show Sequence

>gi|335610|gb|M12882.1|VACENVANT Vaccinia virus (HindIII F fragment) p37K gene, encoding an envelope antigen
GTCGACTTTGATGAAAATTTTAGCGATATAGCCGATGATATTCTAGATCGTTGATAGAACAGGATGTATA
AGTTTTTATGTTAACTAAATGTGGCCATTTGCATCGGTACCTGCGGGAGCAAAATGTAGGCTGGTAGAAA
CACTACCAGAAAATATGGATTTTAGATCCGATCATTTAACAACATTTGAATGTTTTAACGAAATTATCAC
TCTAGCTAAGAAATATATATACATAGCATCTTTTTGTTGTAATCCTCTGAGTACGACTAGGGGAGCGCTT
ATTTTTGATAAACTAAAAGAGGCATCTGAAAAAGGGATTAAAATAATAGTTTTGCTAGATGAACGAGGGA
AAAGAAATCTGGGAGAGCTACAAAGTCACTGCCCGGATATAAATTTTATAACCGTTAATATAGATAAAAA
AAATAATGTGGGACTACTACTCGGTTGTTTTTGGGTGTCAGATGATGAAAGATGTTATGTAGGAAACGCG
TCATTTACTGGAGGATCTATACATACGATTAAAACGTTAGGTGTATATTCTGATTATCCCCCGCTGGCCA
CAGATCTTCGTAGAAGATTTGATACTTTTAAAGCCTTTAATAGCGCAAAAAATTCATGGTTGAATTTATG
CTCTGCGGCTTGTTGTTTGCCAGTTAGCACTGCGTATCATATTAAGAATCCTATAGGTGGAGTGTTCTTT
ACTGATTCTCCGGAACACCTATTGGGATATTCTAGAGATCTAGATACCGATGTAGTTATTGATAAACTCA
AGTCGGCTAAGACTAGTATAGATATTGAACATTTGGCCATAGTTCCCACTACACGTGTCGACGGTAATAG
CTACTATTGGCCCGACATTTACAACTCCATTATAGAAGCAGCCATTAATAGAGGAGTTAAGATCAGACTT
CTAGTTGGTAATTGGGATAAGAACGACGTATATTCTATGGCAACCGCCAGAAGTCTAGACGCGTTGTGTG
TTCAAAATGATCTATCTGTGAAGGTTTTCACTATTCAGAATAATACAAAATTGTTGATAGTCGACGACGA
ATATGTTCATATCACTTCGGCAAATTTCGACGGAACCCATTACCAAAATCACGGATTCGTCAGTTTTAAT
AGTATAGATAAACAGCTTGTAAGCGAGGCTAAAAAAATATTTGAGAGAGATTGGGTATCTAGCCACAGTA
AATCGTTAAAAATTTAAAAAAAAGAAAATAGAGACGTATAGA

Protein Sequence : Show Sequence

>gi|335611|gb|AAA48235.1| envelope antigen
MWPFASVPAGAKCRLVETLPENMDFRSDHLTTFECFNEIITLAKKYIYIASFCCNPLSTTRGALIFDKLK
EASEKGIKIIVLLDERGKRNLGELQSHCPDINFITVNIDKKNNVGLLLGCFWVSDDERCYVGNASFTGGS
IHTIKTLGVYSDYPPLATDLRRRFDTFKAFNSAKNSWLNLCSAACCLPVSTAYHIKNPIGGVFFTDSPEH
LLGYSRDLDTDVVIDKLKSAKTSIDIEHLAIVPTTRVDGNSYYWPDIYNSIIEAAINRGVKIRLLVGNWD
KNDVYSMATARSLDALCVQNDLSVKVFTIQNNTKLLIVDDEYVHITSANFDGTHYQNHGFVSFNSIDKQL
VSEAKKIFERDWVSSHSKSLKI

Related Vaccine(s): LC16m8

14. H3L

Gene Name : H3L
Sequence Strain (Species/Organism) : Vaccinia virus
VO ID : VO_0010935
NCBI Gene ID : 3707557
NCBI Protein GI : 66275898
Locus Tag : VACWR101
Genbank Accession : KT184691
Protein Accession : YP_232983
Taxonomy ID : 10245
Gene Starting Position : 88322
Gene Ending Position : 89296
Gene Strand (Orientation) : -
Protein Name : IMV heparin binding surface protein
Protein pI : 6.43
Protein Weight : 34569.08
Protein Length : 324
Protein Note : similar to VACCP-H3L; involved in IMV maturation

DNA Sequence : Show Sequence

>NC_006998.1:88322-89296 Vaccinia virus, complete genome
GTTAGATAAATGCGGTAACGAATGTTCCTGTAAGGAACCATAACAGTTTAGATTTAACGTTAAAGATGAG
CATAAACATAATAAACAAAATTACAATCAAACCTATAACATTAATATCAAACAATCCAAAAAATGAAATC
AGTGGAGTAGTAAACGCGTACATAACTCCTGGATAACGTTTAGTAGCTGCCGTTCCTATTCTAGACCAAA
AATTCGGTTTCATGTTTTCGAAACGGTGTTCTGCAACAAGTCGGGGATCGTGTTCTACATATTTGGCGGC
ATTATCCAGTATCTGCCTATTGATCTTCATTTCGTTTTCAATTCTGGCTATTTCAAAATAAAATCCCGAT
GATAGACCTCCAGACTTTATAATTTCATCTACGATGTTCAGCGCCGTAGTAACTCTAATAATATAGGCTG
ATAAGCTAACATCATACCCTCCTGTATATGTGAATATGGCATGATTTTTGTCCATTACAAGCTCGGTTTT
AACTTTATTGCCTGTAATAATTTCTCTCATCTGTAGGATATCTATTTTTTTGTCATGCATTGCCTTCAAG
ACGGGACGAAGAAACGTAATATCCTCAATAACGTTATCGTTTTCTACAATAACTACATATTCTACCTTTT
TATTTTCTAACTCGGTAAAAAAATTAGAATCCCATAGGGCTAAATGTCTAGCGATATTTCTTTTCGTTTC
CTCTGTACACATAGTGTTACAAAACCCTGAAAAGAAGTGAGTATACTTGTCATCATTTCTAATGTTTCCT
CCAGTCCACTGTATAAACGCATAATCCTTGTAATGATCTGGATCATCCTTGACTACCACAACATTTCTTT
TTTCTGGCATAACTTCGTTGTCCTTTACATCATCGAACTTCTGATCATTAATATGCTCATGAACATTAGG
AAATGTTTCTGATGGAAGTCTATCAATAACTGGCACAACAATAACAGGAGTTTTCGCCGCCGCCA

Protein Sequence : Show Sequence

>YP_232983.1 IMV heparin binding surface protein [Vaccinia virus]
MAAAKTPVIVVPVIDRLPSETFPNVHEHINDQKFDDVKDNEVMPEKRNVVVVKDDPDHYKDYAFIQWTGG
NIRNDDKYTHFFSGFCNTMCTEETKRNIARHLALWDSNFFTELENKKVEYVVIVENDNVIEDITFLRPVL
KAMHDKKIDILQMREIITGNKVKTELVMDKNHAIFTYTGGYDVSLSAYIIRVTTALNIVDEIIKSGGLSS
GFYFEIARIENEMKINRQILDNAAKYVEHDPRLVAEHRFENMKPNFWSRIGTAATKRYPGVMYAFTTPLI
SFFGLFDINVIGLIVILFIMFMLIFNVKSKLLWFLTGTFVTAFI

Molecule Role : Protective antigen
Molecule Role Annotation : Mice were immunized with recombinant H3L protein to examine H3L-specific antibody responses in greater detail. H3L-immunized mice developed high-titer vaccinia virus-neutralizing antibodies (mean PRNT50 = 1:3,760). Importantly, H3L-immunized mice were subsequently protected against lethal intranasal challenges with 1 or 5 50% lethal doses (LD50) of pathogenic vaccinia virus strain WR, demonstrating the in vivo value of an anti-H3L response. To formally demonstrate that neutralizing anti-H3L antibodies are protective in vivo, we performed anti-H3L serum passive-transfer experiments. Mice receiving H3L-neutralizing antiserum were protected from a lethal challenge with 3 LD50 of vaccinia virus strain WR (5/10 versus 0/10; P < 0.02) (Davies et al., 2005).
Related Vaccine(s): IMV-EEV

15. L1R from Monkeypox virus Zaire-96-I-16

Gene Name : L1R from Monkeypox virus Zaire-96-I-16
Sequence Strain (Species/Organism) : Monkeypox virus Zaire-96-I-16
VO ID : VO_0010871
NCBI Gene ID : 928938
NCBI Protein GI : 17974998
Locus Tag : MPXVgp085
Genbank Accession : AF380138
Protein Accession : NP_536512
Taxonomy ID : 619591
Gene Starting Position : 80280
Gene Ending Position : 80738
Gene Strand (Orientation) : +
Protein Name : L1R
Protein pI : 4.99
Protein Weight : 17001.2
Protein Length : 152
Protein Note : similar to Vaccinia virus strain Copenhagen J1R

DNA Sequence : Show Sequence

>NC_003310.1:80280-80738 Monkeypox virus Zaire-96-I-16, complete genome
AATGGATCACAACCAGTATCTCTTAACGATGTTCTTCGCAGATGATGATTCATTTTTTAAGTATTTTGCT
AGTCAAGATGATGAATCTTCATTATCTGATATATTGCAAATCACTCAATATCTAGACTTTCTGTTATTAT
TATTGATCCAATCAAAAAATAAATTAGAAGCTGTGGGTCATTGTTATGAATCTCTTTCAGAGGAATACAG
ACAATTGACAAAATTCACAGACTCTCAAGATTTTAAAAAACTGTTTAACAAGGTCCCTATTGTTACAGAT
GGAAGGGTCAAACTTAATAAAGGATATTTGTTCGACTTTGTGATTAGTTTGATGCGATTCAAAAAAGAAT
CAGCTCTAGCTACCACCGCAATAGATCCTGTTAGATACATAGATCCTCGTCGTGATATCGCATTTTCTAA
CGTGATGGATATATTAAAGTCGAATAAAGTTGAACAATA

Protein Sequence : Show Sequence

>NP_536512.1 L1R [Monkeypox virus Zaire-96-I-16]
MDHNQYLLTMFFADDDSFFKYFASQDDESSLSDILQITQYLDFLLLLLIQSKNKLEAVGHCYESLSEEYR
QLTKFTDSQDFKKLFNKVPIVTDGRVKLNKGYLFDFVISLMRFKKESALATTAIDPVRYIDPRRDIAFSN
VMDILKSNKVEQ

Molecule Role : Protective antigen
Molecule Role Annotation : It has previously been shown that a DNA subunit vaccine (4pox) based on four orthopoxvirus immunogens (L1R, B5R, A27L and A33R) can produce protective immunity against lethal orthopoxvirus challenges in mice and nonhuman primates. the immunogenicity of an L1R construct to which a tissue plasminogen activator signal sequence was placed in frame with the full-length L1R gene was tested. When the tPA-L1R construct was substituted for the unmodified L1R gene in the 4pox vaccine, given as a prime and single boost, animals were better protected from lethal challenge with vaccinia virus (VACV) (Golden et al., 2008).
Related Vaccine(s): Smallpox DNA Vaccine

16. L1R from Vaccinia virus (strain: WR (Western Reserve))

Gene Name : L1R from Vaccinia virus (strain: WR (Western Reserve))
Sequence Strain (Species/Organism) : Vaccinia virus (strain: WR (Western Reserve))
NCBI Gene ID : 3707544
Locus Tag : VACWR088
Gene Strand (Orientation) : ?
Protein Note : IMV membrane protein

DNA Sequence : Show Sequence

>gi|61387|emb|X01978.1| Vaccinia virus gene cluster with thymidine kinase gene
AAGCTTTTAGAATATGTGCATATAGGACCACTAGCAAAAGATAAAGAGGATAAAGTAAAGAAAAGATATC
CAGAGTTTAGATTAGTCAACACAGGACCCGGTGGTCTTTCGGCATTGTTAAGACAATCGTATAATGGAAC
CGCACCCAATTGCTGTCGCACTTTTAATCGTACTCATTATTGGAAGAAGGATGGAAAGATATCAGATAAG
TATGAAGAGGGTGCAGTATTAGAATCGTGTTGGCCAGACGTTCACGACACCGGAAAATGCGATGTTGATT
TATTCGACTGGTGTCAGGGGGATACGTCCGATAGAAACATATGCCATCAGTGGATCGGTTCAGCCTTTAA
TAGGAGTAATAGAACTGTAGAGGGTCAACAATCGTTAATAAATCTGTATAATAAGATGCAAACATTATGT
AGTAAAGATGCTAGTGTACCAATATGTGAATCATTTTTGCATCATTTACGCGCACACAATACAGAAGATA
GCAAAGAGATGATCGATTATATTCTAAGACAACAGTCTGCGGACTTTAAACAGAAATATATGAGATGTAG
TTATCCCACTAGAGATAAGTTAGAAGAGTCATTAAAATATGCGGAACCTCGAGAATGTTGGGATCCAGAG
TGTTCGAATGCCAATGTTAATTTCTTGCTAACACGTAATTATAATAATTTAGGACTTTGCAATATTGTAC
GATGTAATACTAGCGTGAACAACTTACAGATGGATAAAACTTCCTCATTAAGATTGTCATGTGGATTAAG
CAATAGTGATAGATTTTCTACTGTTCCCGTCAATAGAGCAAAAGTAGTTCAACATAATATTAAACACTCG
TTCGACTCAAAATTGCATTTGATCAGTTTATTATCTCTCTTGGTAATATGGATACTAATTGTAGCTATTT
AAATGGGTGCCGCGGCAAGCATACAGACGACGGTGAATACACTCAGCGAACGTATCTCGTCTAAATTAGA
ACAAGAAGCGAATGCTAGTGCTCAAACAAAATGTGATATAGAAATCGGAAATTTTTATATCCGACAAAAC
CATGGATGTAACCTCACTGTTAAAAATATGTGCTCTGCGGACGCGGATGCTCAGTTGGATGCTGTGTTGT
CAGCCGCTACAGAAACATATAGTGGATTAACACCGGAACAAAAAGCATACGTGCCAGCTATGTTTACTGC
TGCGTTAAACATTCAGACGAGTGTAAACACTGTTGTTAGAGATTTTGAAAATTATGTGAAACAGACTTGT
AATTCTAGCGCGGTCGTCGATAACAAATTAAAGATACAAAACGTAATCATAGATGAATGTTACGGAGCCC
CAGGATCTCCAACAAATTTGGAATTTATTAATACAGGATCTAGCAAAGGAAATTGTGCCATTAAGGCGTT
GATGCAATTGACGACTAAGGCCACTACTCAAATAGCACCTAAACAAGTTGCTGGTACAGGAGTTCAGTTT
TATATGATTGTTATCGGTGTTATAATATTGGCAGCGTTGTTTATGTACTATGCCAAGCGTATGTTGTTCA
CATCCACCAATGATAAAATCAAACTTATTTTAGCCAATAAGGAAAACGTCCATTGGACTACTTACATGGA
CACATTCTTTAGAACTTCTCCGATGGTTATTGCTACCACGGATATGCAAAACTGAAAATATATTGATAAT
ATTTTAATAGATTAACATGGAAGTTATCACTGATCGTCTAGACGATATAGTGAAACAAAATATAGCGGAT
GAAAAATTTGTAGATTTTGTTATACACGGTCTAGAGCATCAATGTCCTGCTATACTTCGACCATTAATTA
GGTTGTTTATTGATATACTATTATTTGTTATAGTAATTTATATTTTTACGGTACGTCTAGTAAGTAGAAA
TTATCAAATGTTGTTGGCGTTGGTGGCGCTAGTCATCACAATTAACTATTTTTTATTACTTTATACTATA
ATAGTACTAGACTGACTTCTAACAAACATCTCACCTGCCATAAATAAATGCTTGATATTAAAGTCTTCTA
TTTCTAACACTATTCCATCTGTGGAAAATAATACTCTGACATTATCGCTAATTGACACATCGGTGAGTGA
TATGCCTATAAAGTAATAATCTTCTTTGGGCACATATACCAGTGTACCAGGTTCTAACAACCTATTTACT
GGTGCTCCTATAGCATACTTTTTCTTTACCTTGAGAATATCCATCGTTTGCTTGGTCAATAGCGATATGT
GATTTTTTATCAACCACTCGAAAAAGTAATTGGAGTGTTCATATCCTCTACGGGCTATTGTCTCATGGCC
GTGTATGAAATTTAAGTAACACGACTGTGGTAGATTTGTTCTATAGAGCCGGTTGCCGCAAATAGATAGA
ACTACCAATATGTCTGTACAAATGTTAAACATTAATTGATTAACAGAAAAAACAATGTTCGTTCTGGGAA
TAGAAACCAGATCAAAACAAAATTCGTTAGAATATATGCCACGTTTATACATTGAATATAAAATAACTAC
AGTTTGAAAAATAACAGTATCATTTAAACATTTAACTTGCGGGGTTAATCTCACAACTTTACTGTTTTTG
AACTGTTCAAAATATAGCATAGATCCGTGAGAAATACGTTTAGCCGCCTTTAATAGAGGAAATCCCACCG
CCTTTCTGGATCTCACCAACGACGATAGTTCTGACCAGCAACTCATTTCTTCATCATCCACCTGTTTTAA
CATATAATAGGCAGGAGATAGATATCCGTCATTGCAATATTCCTTCTCGTAGGCACACAATCTAATATTG
ATAAAATCTCCATTCTCTTCTCTGCATTTATTATCTTGTTTCGGTGGCTGATTAGGCTGTAGTCTTGGTT
TAGGCTTTGGTATATCGTTGTTGAATCTATTTTGGTCATTAAATCTTTCATTTCTTCCTGGTATATTTTT
ATCACCTCGTTTGGTTGGATTTTTGTCTATATTATCGTTTGTAACATCGGTACGGGTATTCATTTATCAC
AAAAAAAACTTCTCTAAATGAGTCTACTGCTAGAAAACCTCATCGAAGAAGATACCATATTTTTTGCAGG
AAGTATATCTGAGTATGATGATTTACAAATGGTTATTGCCGGCGCAAAATCCAAATTTCCAAGATCTATG
CTTTCTATTTTTAATATAGTACCTAGAACGATGTCAAAATATGAGTTGGAGTTGATTCATAACGAAAATA
TCACAGGAGCAATGTTTACCACAATGTATAATATAAGAAACAATTTGGGTCTAGGAGATGATAAACTAAC
TATTGAAGCCATTGAAAACTATTTCTTGGATCCTAACAATGAAGTTATGCCTCTTATTATTAATAATACG
GATATGACTGCCGTCATTCCTAAAAAAAGTGGTAGGAGAAAGAATAAGAACATGGTTATCTTCCGTCAAG
GATCATCACCTATCTTGTGTATTTTCGAAACTCGTAAAAAGATTAATATTTATAAAGAAAATATGGAATC
CGCGTCGACTGAGTATACACCTATCGGAGACAACAAGGCTTTGATATCTAAATATGCGGGAATTAATATC
CTAAATGTGTATTCTCCTTCCACATCCATAAGATTGAATGCCATTTACGGATTCACCAATAAAAATAAAC
TAGAGAAACTTAGTACTAATAAGGAACTAGAATCGTATAGTTCTAGCCCTCTTCAAGAACCCATTAGGTT
AAATGATTTTCTGGGACTATTGGAATGTGTTAAAAAGAATATTCCTCTAACAGATATTCCGACAAAGGAT
TGATTACTATAAATGGAGAATGTTCCTAATGTATACTTTAATCCTGTGTTTATAGAGCCCACGTTTAAAC
ATTCTTTATTAAGTGTTTATAAACACAGATTAATAGTTTTATTTGAAGTATTCGTTGTATTCATTCTAAT
ATATGTATTTTTTAGATCTGAATTAAATATGTTCTTCATGCCTAAACGAAAAATACCCGATCCTATTGAT
AGATTACGACGTGCTAATCTAGCGTGTGAAGACGATAAATTAATGATCTATGGATTACCATGGATGACAA
CTCAAACATCTGCGTTATCAATAAATAGTAAACCGATAGTGTATAAAGATTGTGCAAAGCTTTTGCGATC
AATAAATGGATCACAACCAGTATCTCTTAACGATGTTCTTCGCAGATGATGATTCATTTTTTAAGTATTT
GGCTAGTCAAGATGATGAATCTTCATTATCTGATATATTGCAAATCACTCAATATCTAGACTTTCTGTTA
TTATTATTGATCCAATCAAAAAATAAATTAGAAGCCGTGGGTCATTGTTATGAATCTCTTTCAGAGGAAT
ACAGACAATTGACAAAATTCACAGACTTTCAAGATTTTAAAAAACTGTTTAACAAGGTCCCTATTGTTAC
AGATGGAAGGGTCAAACTTAATAAAGGATATTTGTTCGACTTTGTGATTAGTTTGATGCGATTCAAAAAA
GAATCCTCTCTAGCTACCACCGCAATAGATCCTGTTAGATACATAGATCCTCGTCGCAATATCGCATTTT
CTAACGTGATGGATATATTAAAGTCGAATAAAGTGAACAATAATTAATTCTTTATTGTCATCATGAACGG
CGGACATATTCAGTTGATAATCGGCCCCATGTTTTCAGGTAAAAGTACAGAATTAATTAGACGAGTTAGA
CGTTATCAAATAGCTCAATATAAATGCGTGACTATAAAATATTCTAACGATAATAGATACGGAACGGGAC
TATGGACGCATGATAAGAATAATTTTGAAGCATTGGAAGCAACTAAACTATGTGATGTCTTGGAATCAAT
TACAGATTTCTCCGTGATAGGTATCGATGAAGGACAGTTCTTTCCAGACATTGTTGAATTCTGTGAGCGT
ATGGCAAACGAAGGAAAAATAGTTATAGTAGCCGCACTCGATGGGACATTTCAACGTAAACCGTTTAATA
ATATTTTGAATCTTATTCCATTATCTGAAATGGTGGTAAAACTAACTGCTGTGTGTATGAAATGCTTTAA
GGAGGCTTCCTTTTCTAAACGATTGGGTGAGGAAACCGAGATAGAAATAATAGGAGGTAATGATATGTAT
CAATCGGTGTGTAGAAAGTGTTACATCGACTCATAATATTATATTTTTTATCTAAAAAACTAAAAATAAA
CATTGATTAAATTTTAATATAATACTTAAAAATGGATGTTGTGTCGTTAGATAAACCGTTTATGTATTTT
GAGGAAATTGATAATGAGTTAGATTACGAACCAGAAAGTGCAAATGAGGTCGCAAAAAAACTGCCGTATC
AAGGACAGTTAAAACTATTACTAGGAGAATTATTTTTTCTTAGTAAGTTACAGCGACACGGTATATTAGA
TGGTGCCACCGTAGTGTATATAGGATCTGCTCCCGGTACACATATACGTTATTTGAGAGATCATTTCTAT
AATTTAGGAGTGATCATCAAATGGATGCTAATTGACGGCCGCCATCATGATCCTATTTTAAATGGATTGC
GTGATGTGACTCTAGTGACTCGGTTCGTTGATGAGGAATATCTACGATCCATCAAAAAACAACTGCATCC
TTCTAAGATTATTTTAATTTCTGATGTGAGATCCAAACGAGGAGGAAATGAACCTAGTACGGCGGATTTA
CTAAGTAATTACGCTCTACAAAATGTCATGATTAGTATTTTAAACCCCGTGGCGTCTAGTCTTAAATGGA
GATGCCCGTTTCCAGATCAATGGATCAAGGACTTTTATATCCCACACGGTAATAAAATGTTACAACCTTT
TGCTCCTTCATATTCAGCTGAAATGAGATTATTAAGTATTTATACCGGTGAGAACATGAGACTGACTCGA
GTTACCAAATCAGACGCTGTAAATTATGAAAAAAAGATGTACTACCTTAATAAGATCGTCCGTAACAAAG
TAGTTGTTAACTTTGATTATCCTAATCAGGAATATGACTATTTTCACATGTACTTTATGCTGAGGACCGT
GTACTGCAATAAAACATTTCCTACTACTAAAGCAAAGGTACTATTTCTACAACAATCTATATTTCGTTTC
TTAAATATTCCAACAACATCAACTGAAAAAGTTAGTCATGAACCAATACAACGTAAAATATCTAGCAAAA
ATTCTATGTCTAAAAACAGAAATAGCAAGAGATCCGTACGCAGTAATAAATAGAAACGTACTACTGAGAT
ATACTACCGATATAGAGTATAATGATTTAGTTACTTTAATAACCGTTAGACATAAAATTGATTCTATGAA
AACTGTGTTTCAGGTATTTAACGAATCATCCATAAATTATACTCCGGTTGATGATGATTATGGAGAACCA
ATCATTATAACATCGTATCTTCAAAAAGGTCATAACAAGTTTCCTGTAAATTTTCTATACATAGATGTGG
TAATATCTGACTTATTTCCTAGCTTTGTTAGACTAGATACTACAGAAACTAATATAGTTAATAGTGTACT
ACAAACAGGCGATGGTAAAAAGACTCTTCGTCTTCCCAAAATGTTAGAGACGGAAATAGTTGTCAAGATT
CTCTATCGCCCTAATATACCATTAAAAATTGTTAGATTTTTCCGCAATAACATGGTAACTGGAGTAGAGA
TAGCCGATAGATCTGTTATTTCAGTCGCTGATTAATCAATTAGTAGAGATGAGATAAGAACATTATAATA
ATCAATAATATATCTTATATCTCGTTTAGAAAAATGCTAATATTAAAATAGCTAACGCAGTAATCCAATC
GGAAGCCATTTGATATCTATAATAGGGTATCTAATTTCCTGATTCAGATAGCGGACAGCTATATTCTCGG
TAGCTACTCGTTTGGAATCACAAACATTATTTACATCTAATTTACTATCTGTAATGGAAACGTTTCCCAA
TGAAATGGTACAATCCGATACATTGCATTTTGTTATATTTTTTTTTAAAGAGGCTGGTAACAACGCATCG
CTTCGTTTACATGGCTCGTACCAACAATAATAGGGTAATCTTGTATCTATTCCTATCCGTACTATGCTTT
TATCAGGATAAATACATTTACATCGTATATCGTCTTTGTTAGCATCACAGAATGCATAAATTTGTTCGTC
CGTCATGATAAAAATTTAAAGTGTAAATATAACTATTATTTTTATAGTTGTAATAAAAAGGGAAATTTGA
TTGTATACCTTCGGTTCTTTAAAAGAAACTGACTTGATAAAAATGGCTGTAATCTCTAAGGTTACGTATA
GTCTATATGATCAAAAAGAGATTAATGCTACAGATATTATCATTAGTCATGTTAAAAATGACGACGATAT
CGGTACCGTTAAAGATGGTAGACTAGGTGCTATGGATGGGGCATTATGTAAGACTTGTGGGAAAACGGAA
TTGGAATGTTTCGGTCAGTGGGGTAAAGTAAGTATTTATAAAACTCATATAGTTAAGCCTGAATTTATTT
CAGAAATCACTCGTTTACTGAATCATATATGTATTCACTGCGGATTATTGCGTTCACGAGAACCGTATTC
CGACGATATTAACCTAAAAGAGTTAT

Protein Sequence : Show Sequence

>gi|61389|emb|CAA26010.1| F2 polypeptide [Vaccinia virus]
MGAAASIQTTVNTLSERISSKLEQEANASAQTKCDIEIGNFYIRQNHGCNLTVKNMCSADADAQLDAVLS
AATETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENYVKQTCNSSAVVDNKLKIQNVIIDECYGAP
GSPTNLEFINTGSSKGNCAIKALMQLTTKATTQIAPKQVAGTGVQFYMIVIGVIILAALFMYYAKRMLFT
STNDKIKLILANKENVHWTTYMDTFFRTSPMVIATTDMQN

Related Vaccine(s): IMV-EEV

17. p53

Gene Name : p53
Sequence Strain (Species/Organism) : Homo sapiens
NCBI Gene ID : 7157
Other Database IDs : TP53, LFS1, TRP53, tumor protein p53 (Li-Fraumeni syndrome)

DNA Sequence : Show Sequence

>gi|42821409|dbj|AB118156.1| Homo sapiens p53 gene for P53, exon 5, partial cds
TACTCCCCTGCCCTCAACAAGATGTTTTGCCAACTGGCCAAGACCTGCCCTGTGCAGCTGTGGGTTGATT
CCACACCCCCGCCCGGCACCCGCGTCCGCGCCATGGCCATCTACAAGCAGTCACAGCACATGACGGAGGT
TGTGAGGCGCTGCCCCCACCATGAGCGCTGCTCAGATAGCGATG

Protein Sequence : Show Sequence

>gi|42821410|dbj|BAD11806.1| P53 [Homo sapiens]
YSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSD

Related Vaccine(s): dVV-L

18. VACVgp196

Gene Name : VACVgp196
Sequence Strain (Species/Organism) : Vaccinia virus
NCBI Gene ID : 1486323
Locus Tag : VACVgp196
Gene Strand (Orientation) : ?
Protein Note : EEV glycoprotein

Protein Sequence : Show Sequence

>gi|335508|gb|AAA48160.1| putative A33R [Vaccinia virus Copenhagen]
MMTPENDEEQTSVFSATVYGDKIQGKNKRKRVIGLCIRISMVISLLSMITMSAFLIVRLNQCMSANEAAI
TDAAVAVAAASSTHRKVASSTTQYDHKESCNGLYYQGSCYILHSDYQLFSDAKANCTAESSTLPNKSDVL
ITWLIDYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCVKTMN

Related Vaccine(s): IMV-EEV

19. VACVgp200

Gene Name : VACVgp200
Sequence Strain (Species/Organism) : Vaccinia virus
NCBI Gene ID : 1486327
Locus Tag : VACVgp200
Gene Strand (Orientation) : ?
Protein Note : EEV membrane protein

Protein Sequence : Show Sequence

>gi|335512|gb|AAA48164.1| putative A36R [Vaccinia virus Copenhagen]
MMLVPLITVTVVAGTILVCYILYICRKKIRTVYNDNKIIMTKLKKIKSSNSSKSSKSTDSESDWEDHCSA
MEQNNDVDNISRNEILDDDSFAGSLIWDNESNVMAPSTEHIYDSVAGSTLLINNDRNEQTIYQNTTVVIN
ETETVEVLNEDTKQNPNYSSNPFVNYNKTSICSKSNPFITELNNKFSENNPFRRAHSDDYLNKQEQDHEH
DDIESSVVSLV

Related Vaccine(s): IMV-EEV

III. Vaccine Information

1. ACAM2000

a. Product Name:

Smallpox (Vaccinia) Vaccine, Live

b. Tradename:

ACAM2000

c. Manufacturer:

Acambis, Inc

d. Vaccine Ontology ID:

VO_0000003

e. CDC CVX code:

f. Type:

Replication competent virus

g. Status:

Licensed

h. Location Licensed:

USA (License #1733)

i. Host Species for Licensed Use:

Human

j. Allergen:

Neomycin, Polymyxin B

k. Preparation

ACAM2000 was prepared from ACAM1000 master seed stock and produced in Vero cells to address the need for rapid large-scale vaccine production (Parrino et al., 2006).
Specifically, ACAM2000 was manufactured by infecting Vero cells grown on microcarriers under serum-free conditions with the P9 production virus inoculum at an MOI of 0.01–0.2. Virus particles were purified and concentrated. The resulting concentrated bulk vaccine was formulated by dilution with a buffer containing stabilizers to a final potency of 1.0–5.0 × 10^8 pfu/mL, filled into vials containing 0.3 mL (Monath et al., 2004).

l. Immunization Route

Percutaneous

m. Virulence

It has long been known that vaccinia strains differ with respect to neurovirulence in infant mice. Clones 1, 3, and 5 and the uncloned virus had virulence properties that were unacceptable for consideration as vaccine candidates. The same four viruses that had exhibited excessive virulence in rabbit skin were significantly more neurovirulent than Dryvax1 (p < 0.05, Kaplan—Meier survival distribution, log rank test), whereas clones 2, 4, and 6 were similar to Dryvax1 or less virulent. The more virulent viruses also replicated to higher titer in mouse brain. In these initial experiments Clone 2 did not appear to be attenuated with respect to neurovirulence, but subsequent studies with larger numbers of animals showed significantly higher survival distribution compared to Dryvax1. Clone 2 (renamed ACAM1000) was selected as the candidate for further development, based on its similarity to Dryvax1 in pock formation in rabbit skin but its lower neurovirulence in mice and monkeys. Seed viruses and vaccine produced from each bioreactor run were tested for neurovirulence in suckling mice, using Dryvax1 as a comparator. Plaque-purified vaccinia virus lines were shown to differ significantly in neurovirulence for mice, in their ability to evoke immune responses against the inserted gene product, and in their HindIII restriction maps. The variant viruses often exhibit reduced infectivity and reduced virulence for mice. We found biological and molecular heterogeneity among 6 clones derived from Dryvax1, with some clonal subpopulations (e.g. Clone 3) having dramatically higher virulence and changes at the genomic level. The degree of neurovirulence for suckling mice was used to distinguish vaccine strains with low, moderate, or high pathogenicity (Vilesova et al., 1985). The new vaccine has advantages over first generation vaccines, since it has been produced to modern manufacturing and control standards, is free from adventitious agents , and does not contain subpopulations of virus with undesirable virulence properties (Monath et al., 2004).
In addition, mice immunized with MVA were protected against lethal infection with a more virulent form of vaccinia virus altered to coexpress IL-4. IL-4 diminishes the cytolytic capacity of CD81 T cells, resulting in delayed viral clearance and increased virulence (Parrino et al., 2006).

n. Storage

After reconstitution, ACAM2000 vaccine may be administered within 6 to 8 hours if kept at room temperature (20-25°C, 68-77°F); it should then be discarded as a biohazardous material. Unused, reconstituted ACAM2000 vaccine may be stored in a refrigerator (2-8°C, 36-46°F) up to 30 days, after which it should be discarded as a biohazardous material (FDA: ACAM2000).

o . Approved Age for Licensed Use

1 year of age and older

p. Contraindication

Individuals with severe immunodeficiency who are not expected to benefit from the vaccine (FDA: ACAM2000).

q. Description

The benefits of cloning appeared to outweigh the recognized risk that a clonal virus population may differ biologically from the ‘genetic swarm’ represented by the animal-skin vaccine. Because it would not be possible to conduct field tests for efficacy, the new vaccine would need to match the licensed vaccine (Dryvax®) as closely as possible in preclinical tests for safety, immunogenicity, and protective activity and in clinical trials for safety and immunogenicity (Monath et al., 2004).
Clinical trials have been conducted using the NYCBH-derived ACAM2000 vaccinia virus-based vaccine. On the basis of animal studies, ACAM2000 is believed to be less neurovirulent than Dryvax. ACAM2000 was similar to Dryvax in its ability to induce immune responses and in reactogenicity in phase I trials. During phase II and phase III clinical trials, cases of myopericarditis were associated with both ACAM2000 and Dryvax in vaccinia-naive volunteers (Parrino et al., 2006).

r. Mouse Response

Host Strain: 3-4 day-old outbred ICR mice
Vaccination Protocol: Groups of mice were inoculated with graded doses (0.3 to 3.0 log10 pfu) (Monath et al., 2004).
Persistence: Survival analysis showed that ACAM1000 and ACAM2000 did not differ from one another but had significantly longer survival than Dryvax (Monath et al., 2004).
Side Effects: We showed that ACAM1000 and ACAM2000 were significantly less neurovirulent for mice and monkeys than the parental Dryvax1 virus, presumably. ACAM2000 should be less likely to cause post-vaccinal encephalitis in humans. However, the pathogenesis of postvaccinal
encephalitis is still uncertain. Vaccinia virus has been isolated from CSF and brain, suggesting that the virus invades the central nervous system in humans (Monath et al., 2004).
Efficacy: The median lethal dose (LD50) and 90% lethal dose (LD90) were higher for mice receiving ACAM2000 and ACAM1000 compared to Dryvax (Monath et al., 2004).
Description: The neurovirulence profiles of ACAM2000 and ACAM1000 vaccines were compared to Dryvax in a lethal dose assay (Monath et al., 2004).

s. Mouse Response

Host Strain: Young adult BALB/c mice.
Vaccination Protocol: Groups of 5 mice were immunized with graded doses (4 to 7 log10 PFU/mL) of ACAM 2000 and then challenged 3 weeks later with 100 LD50 of vaccinia WR virus. Survival and body weight were recorded daily for 14 days after challenge (Monath et al., 2004).
Persistence: The survival times were not statistically different between treatment groups (Monath et al., 2004).
Side Effects: We showed that ACAM1000 and ACAM2000 were significantly less neurovirulent for mice and monkeys than the parental Dryvax1 virus, presumably. ACAM2000 should be less likely to cause post-vaccinal encephalitis in humans. However, the pathogenesis of postvaccinal
encephalitis is still uncertain. Vaccinia virus has been isolated from CSF and brain, suggesting that the virus invades the central nervous system in humans (Monath et al., 2004).
Efficacy: Protective efficacy of the 3 viruses tested was similar (Monath et al., 2004).
Description: Mice were used to compare the protective efficacy of immunization with ACAM2000, ACAM1000, and Dryvax (Monath et al., 2004).

t. Human Response

Host Strain: Healthy adults aged 18–29 years.
Vaccination Protocol: Clinical development of ACAM2000 commenced with a Phase 1 open-label trial in 100 healthy adults without prior smallpox vaccination. The primary endpoint was the proportion of subjects with a major cutaneous reaction assessed at any time-point from Day 7 (±2) through Day 15 (±2). Fifty-six percent of subjects were male. The majority (89%) were Caucasian; the remaining subjects were African-American (7%), Asian (3%), or Hispanic (1%). The mean age was 23 years, with a range of 18 to 29 years (Monath et al., 2004).
Persistence: Of the 99 subjects who experienced a major cutaneous reaction, 9% had a major cutaneous reaction by Day 3, and the rest experienced a major cutaneous reaction by Day 7. The progression of the cutaneous reaction and its size and appearance were similar to those observed in the trials of ACAM1000. The great majority (96%) developed ≥four fold increases in neutralizing antibodies. The geometric mean neutralizing antibody titer on Day 30 was 225. Four (4%) of 100 subjects did not have a four-fold increase in neutralizing antibody titer on Day 30. However, these 4 subjects all had a major cutaneous reaction by Day 7 (Monath et al., 2004)
Side Effects: With the diminishing threat of smallpox and increased focus on adverse events, vaccination in the United States was discontinued in 1972 for the general public and in 1989 for military personnel. The safety of ACAM2000 was assessed by documentation of adverse events, physical examination findings, lymph node assessments, measurements of vital signs, and clinical laboratory tests, including hematology, clinical chemistry, and urinalysis. Subjects in the study kept a diary of adverse events and took daily oral temperatures. There were no serious adverse events. All 70 subjects (100%) experienced at least one treatment-emergent, expected adverse event during the study. The adverse events were generally mild and did not interfere with the subjects’ daily activities. One subject experienced a serious adverse event, a single new onset seizure on Day 8; this event was considered by the investigator to be remotely related to the study vaccine. The most commonly reported treatment-emergent adverse events were related to the vaccination site and associated lymphadenitis, and the majority of adverse events reported were assessed as mild or moderate in intensity. Elevated temperature was reported as an adverse event for 9 (9%) subjects. Fortunately, cardiac adverse events appear to be self-limiting (Monath et al., 2004).
Efficacy: Ninety-nine percent of the subjects experienced a successful vaccination (Monath et al., 2004).
Description: Phase 1 clinical trials of ACAM1000 and 2000 indicate that the original goal of producing a second generation vaccine that closely matched the safety and immunogenicity of calf-skin vaccine (Dryvax®) was met. The cutaneous, antibody, and T cell responses in primary vaccinees were similar to those elicited by Dryvax®. The appearance and size of the cutaneous lesion and pattern of virus shedding from the vaccination site were also similar. Phase 2 trials in naïve and previously vaccinated subjects have been completed to define the dose response, and to extend safety and immunogenicity data. Phase 3 clinical trials are in progress (Monath et al., 2004).

2. ACAM3000

a. Product Name:

Smallpox (Vaccinia) Vaccine, Live

b. Manufacturer:

ACAMBIS, Inc

c. Type:

Replication competent virus

d. Status:

Licensed

e. Location Licensed:

Cambridge, MA

f. Host Species for Licensed Use:

Human

g. Preparation

ACAM3000 was formulated with 20mM Tris, 0.9% NaCl USP, and 0.01% neomycin USP with a titer of 2.54 × 108 TCID50/mL. The vaccine was reconstituted with 0.9% NaCl and diluted to the appropriate dose. Sterile saline, 0.9% NaCl, was used as the placebo. The dose of MVA administered was verified by back titration for each dose tier that was studied.(Wilck et al., 2010)

h. Immunization Route

Intramuscular injection (i.m.)

3. CCSV

a. Vaccine Ontology ID:

VO_0004090

b. Type:

Replication competent virus

c. Preparation

CCSV was derived from Connaught Laboratories Master Seed number 17633 (originating from the New York City Board of Health vaccinia strain), adapted to replicate in MRC-5 cells, and plaque-purified three times. Cells were infected in ten-layer Nunc cell factories, incubated at 37°C for 3 days, and harvested by trypsinization. Infected cells were sonicated to release intracellular virus. The crude virus bulk was purified and concentrated by ultracentrifugation through a 36% sucrose cushion, and the resulting virus pellet was resuspended in 1 mmol/L Tris buffer (pH 9·0). The undiluted final vaccine material was formulated in 2% human serum albumin to give a concentration of 1×10^8 pfu per mL and was subsequently lyophilised. Lyophilised vials were stored at –20°C before use, reconstituted with 50% glycerin and 0·25% phenol in sterile water for injection, and used within 24 h after dilution (Greenberg et al., 2005).

d. Virulence

(Greenberg et al., 2005)

e. Description

Cell-cultured smallpox vaccine (CCSV) is a replication-competent vaccinia virus vaccine derived from a master seed stock originating from the NYCBH strain. In 2002, a phase I clinical trial conducted in 350 healthy vaccinia-naive and vaccinia-immune adults evaluated the safety, reactogenicity, and immunogenicity of CCSV and Dryvax. Among the study groups, 100 volunteers were assigned to receive various dilutions of CCSV. There were no statistically significant differences between the CCSV and Dryvax groups comparing humoral and cellular immune responses and rates of adverse events. At a delivered dose 50 times lower than the approved Dryvax dose, CCSV was still immunogenic and had a take rate of 100% (Parrino et al., 2006).

f. Human Response

Host Strain: Healthy adults age 18-65 years
Vaccination Protocol: The study was a randomized, blind single-center comparative trial in healthy adult volunteers. Cohorts 1-4 were randomly assigned equivalent doses (2·5×105 plaque-forming units [pfu]) of either CCSV or Dryvax in a double-blind fashion. Participants were stratified by previous exposure to vaccinia (naive vs non-naive) and randomly assigned to vaccine group with a computer-generated process. In the vaccinia-naive group, a ratio of 2 to 1 (CCSV to Dryvax) was used, whereas in the non-naive population, the ratio was 1 to 1. All cohorts were enrolled consecutively with at least a 21-day delay between vaccination of successive cohorts. Cohorts 1-3 consisted of 15, 45, and 90 vaccinia-naive individuals, respectively (100 assigned CCSV and 50 Dryvax). Cohort four consisted of 100 non-naive individuals (50 CCSV and 50 Dryvax). Doses in cohort five (vaccinia-naive) were single-blind (to volunteer only) to one of the following five dilutions of CCSV (20 per group, CCSV to diluent): undiluted, 1:5, 1:10, 1:25, and 1:50. A random subset of 60 volunteers from cohort three (40 CCSV and 20 Dryvax) and 40 volunteers from cohort four (20 from each group) had blood samples taken for testing of cell-mediated immune responses (Greenberg et al., 2005).
Persistence: Participants kept a daily diary of symptoms and body temperature for the first 2 weeks after vaccination, and returned to the clinic for follow-up on days 3, 6, 8, 10, 14, 28, 45, 60, and 180 after vaccination. In vaccinia-naive individuals, titres peaked on day 28, whereas in non-naive people, they peaked on day 14. Although PRN50 geometric mean titres were generally higher for recipients of Dryvax rather than CCSV, their overall patterns on days 14, 28, 60, and 180 after vaccination did not differ significantly between the two vaccine groups for either vaccinia-naive or non-naive individuals (Greenberg et al., 2005).
Side Effects: 349 (99·7%) of 350 volunteers developed pock lesions; one vaccinia-naive individual who received a 1 in 25 dilution of CCSV did not. The rate of adverse events related to vaccine and the extent of humoral and cellular immune responses did not differ between the vaccine groups in vaccinia-naive or non-naive people. During clinic visits in the first 28 days, individuals were assessed for adverse events (vital signs, diary inspection, and concomitant medication) and formation of pock lesions (pock lesion inspection, measurements, and photographs). Intensity of adverse events was classified as mild (awareness of signs and symptoms that are easily tolerated), moderate (signs and symptoms produce discomfort sufficient to interfere with, but not prevent, normal daily activities), or severe (signs and symptoms produce sufficient discomfort to prevent normal daily activities). Differences between two proportions (eg, proportion with adverse events or proportion testing positive by an immunological assay). No serious vaccine-related adverse events were reported. Nobody withdrew from the study because of an adverse event. Other than rashes, no notable differences in frequency or severity of adverse events were recorded in the group receiving undiluted CCSV compared with those receiving diluted CCSV, and there were no notable differences in frequency of adverse events in the 1 in 50 group compared with those in other dilution groups. The adverse events reported for both vaccines were similar in severity and frequency, indicating that the manufacturing process in early human testing did not select for a more reactogenic vaccine, although it never entered larger clinical trials. As expected, vaccinia-non-naive participants tended to have fewer and milder adverse events than their vaccinia-naive counterparts. Overall, the adverse events in the diluted cohort were consistent with those of the other vaccinia-naive cohorts (Greenberg et al., 2005).
Efficacy: The take rate was 100% for all volunteers who received undiluted CCSV, irrespective of previous vaccinia-exposure. The 95% CI of the point estimate for vaccinia-naive individuals was 96–100% for CCSV; similarly, the 95% CI for vaccinia-non-naive individuals was 93%–100%. CCSV was immunogenic in vaccine-naive volunteers at a dose 50 times lower than that approved for Dryvax. CCSV seems to be a safe and immunogenic alternative to calf-lymph derived vaccine for both vaccinia-naive and non-naive people (Greenberg et al., 2005).
Description: U.S. government organizations have identified the need for a new smallpox vaccine to replenish limited stocks of the approved calf-lymph derived vaccine. Previous manufacturing methods using calf lymph are no longer acceptable because of the absence of controls in the process and the potential risk of contamination with the infectious agent associated with the prion disease bovine spongiform encephalitis. New manufacturing methods will need to eliminate the bovine intermediary. Because of ethical and safety considerations, challenge studies or field trials cannot be done to show efficacy. The strategy in designing the cell-cultured smallpox vaccine (CCSV) entailed selection of a well characterised isolate from a master vaccine seed stock used in the WHO eradication campaign. Methods of manufacture included consistency-validated processes for all aspects of manufacture, purification, storage, and distribution. Advantages of manufacture of this vaccine include the breadth of previous experience with well defined human diploid fetal lung fibroblasts in the production of other live, viral human vaccines, and the fact that the process is free from antimicrobial compounds that could produce reactions in sensitised individuals. The aims of this phase 1 clinical trial were to assess safety (frequency and severity of local and systemic adverse events), reactogenicity (frequency and characteristics of pock lesions), and immunogenicity (humoral and cellular immunity assays) of equivalent doses of CCSV and Dryvax in both vaccinia-naive and non-naive, healthy adult volunteers. Additionally, CCSV doses up to 50 times more dilute than the recommended dose for the Dryvax were assessed in a vaccinia-naive population (Greenberg et al., 2005).

4. CJ-50300

a. Type:

Live, attenuated vaccine

b. Status:

Licensed

c. Host Species for Licensed Use:

Human

d. Preparation

CJ-50300 was derived from vaccinia virus strain ATCC VR-118 (originating from the New York City Board of Health vaccinia strain), adapted to replicate in MRC-5 cells under serum-free conditions. Harvested cells were lysed, centrifugated and filtrated. The final bulk was aseptically filled in glass vials and freeze-dried. (Kim et al., 2013)

e. Immunization Route

Intramuscular injection (i.m.)

f. Virulence

Freeze-dried CJ-50300 was reconstituted with 0.3mL glycerin–phenol diluent and used within 1 hour. (Kim et al., 2013)

g. Description

CJ-50300 was derived from vaccinia virus strain ATCC VR-118 adapted to replicate in MRC-5 cells. (Kim et al., 2013)

5. dVV-L

a. Vaccine Ontology ID:

VO_0004094

b. Type:

Live, attenuated vaccine

c. Gene Engineering of p53

Type: Protein
Description: Tumor suppressor protein p53, a nuclear protein, plays an essential role in the regulation of cell cycle, specifically in the transition from G0 to G1. It is found in very low levels in normal cells; however, in a variety of transformed cell lines, it is expressed in high amounts and is believed to contribute to transformation and malignancy. p53 is a DNA-binding protein containing DNA-binding, oligomerization, and transcription activation domains. It is postulated to bind as a tetramer to a p53-binding site and activate expression of downstream genes that inhibit growth and/or invasion and thus function as a tumor suppressor. Mutants of p53 that frequently occur in a number of different human cancers fail to bind the consensus DNA binding site and hence cause the loss of tumor suppressor activity. Alterations of the TP53 gene occur not only as somatic mutations in human malignancies, but also as germline mutations in some cancer-prone families with Li-Fraumeni syndrome (Ober et al., 2002).
Detailed Gene Information: Click here.

d. Adjuvant:

VO ID: VO_0000139
Description: Complete Freund's adjuvant is used during innoculation, followed by a boost in incomplete Freund's adjuvant (Ober et al., 2002)

e. Adjuvant:

VO ID: VO_0000142
Description: Complete Freund's adjuvant is used during innoculation, followed by a boost in incomplete Freund's adjuvant (Ober et al., 2002)

f. Preparation

This vaccine strain was created from the Lister strain by deleting a gene necessary to encode the UDG enzyme, which is essential for a complete cycle of viral replication (Parrino et al., 2006).

g. Virulence

The vaccinia virus strain NYVAC was genetically attenuated by deletion of many nonessential genes, including virulence and host range genes, resulting in a strain growing only in primary cells. Passaging in mammalian cells increases virulence in mammals, resulting in new MVA-like strains with unknown safety profiles in humans. The resulting viruses grow exclusively in a complementing permanent cell line, excluding reversion to virulence and obviating the need for primary cells. The WR strain is a vaccinia virus laboratory strain passaged in mouse brain that has unfavorable properties, such as neurovirulence and gonadotropism, not suitable for clinical use. The large deletions characteristic for MVA seemed to suggest that the restriction in host range and virulence was mainly due to these deletions, including the loss of a host range gene and many immune modulatory genes. It will be interesting to see whether an MVA strain first adapted to growth in mammalian cells and then passaged in mouse brain also regains virulence. In contrast to MVA, dVVs with an essential gene deleted cannot regain replication and virulence functions upon passaging in a chosen host. Reversion to virulence can principally be excluded because the vector lacks an essential gene, which restricts its host range to a complementing cell line (Ober et al., 2002).

h. Description

dVV-L has been evaluated as a poxvirus vaccine. One great advantage of this approach is that the attenuated virus can be manufactured in a cell line that complements the uracil-DNA-glycosylase (UDG) deficiency, rather than in primary cells or eggs as is often needed for other replication-defective viruses, resulting in an improved safety profile and increased capacity for rapid production (Parrino et al., 2006).

i. Mouse Response

Host Strain: Mice (BALB/c/SCID, 6 to 8 weeks old).
Vaccination Protocol: Groups of four immunodeficient mice were challenged subcutaneously (s.c.) with high doses of the wild-type Lister strain or the nonreplicating Lister- or MVA-based vectors (Ober et al., 2002).
Persistence: Using the wild-type Lister virus, doses of ≥10^6 PFU led to a progressive vaccinia virus infection within a 2-month observation period (Ober et al., 2002).
Side Effects: The defective Lister strain-based viruses did not induce any signs of progressive disease. The nonreplicating virus was tolerated without any visible signs of discomfort of the mice at doses of 10^7 and 10^8 PFU. The highest dose of 10^9 PFU was accompanied by mild signs of sickness in the first few days, which disappeared later. At 4 weeks after challenge a lesion was observed at the injection site, which subsequently healed. In summary, not only in the in vitro system but also in vivo in immunodeficient animals, dVVs based on the Lister strain are as well tolerated as the MVA-based viruses (Ober et al., 2002).
Efficacy: A more suitable smallpox prevaccine for immunocompromised subjects, dVV was highly protective in a preclinical challenge model, induced antibodies and CTLs similarly to MVA, and was as safe as MVA-based recombinants in immunodeficient mice. In addition, reversion to virulence can principally be excluded because the vector lacks an essential gene, which restricts its host range to a complementing cell line (Ober et al., 2002).
Description: The main concern about the use of replication-competent viruses in immunotherapy is that severe adverse effects may occur in immunocompromised patients. The mouse model was used to more thoroughly address the safety question in an in vivo model (Ober et al., 2002).

6. IMV-EEV

a. Vaccine Ontology ID:

VO_0004095

b. Type:

Subunit vaccine

c. Gene Engineering of A33R from Vaccinia virus (strain: WR (Western Reserve))

Type: Protein
Description: Similar to VACCP-A33R; associates with A36R; involved in CEV-cell adherence and actin tail formation (NCBI).
Detailed Gene Information: Click here.

d. Gene Engineering of A34R

Type: Protein
Description: Similar to VACCP-A34R; involved in CEV cell adherence and actin tail formation (NCBI).
Detailed Gene Information: Click here.

e. Gene Engineering of A36R

Type: Protein
Description: Similar to VACCP-A36R; interacts with A33R and used in actin tail formation (NCBI).
Detailed Gene Information: Click here.

f. Gene Engineering of B5R from Vaccinia virus (strain: WR (Western Reserve))

Type: Protein
Description: Similar to VACCP-B5R; required for trans-Golgi/endosomal membrane-wrapping of IMV (NCBI).
Detailed Gene Information: Click here.

g. Gene Engineering of D8L

Type: Protein
Description: Infectious intracellular mature virions (IMV), containing a complex core structure and an outer membrane with nonglycosylated viral proteins, are assembled in factory regions within the cytoplasm of vaccinia virus-infected cells. Some IMV migrate out of the factories, become wrapped with an additional double membrane containing viral glycoproteins, and are then transported on microtubules to the periphery of the cell. The outer of the two added membranes fuses with the plasma membrane during exocytosis, and the resulting extracellular particles consist of an IMV surrounded by one extra fragile membrane (Fogg et al., 2004).
Detailed Gene Information: Click here.

h. Gene Engineering of H3L

Type: Protein
Description: Similar to VACCP-H3L; involved in IMV maturation (NCBI).
Detailed Gene Information: Click here.

i. Gene Engineering of L1R from Vaccinia virus (strain: WR (Western Reserve))

Type: Protein
Description: Similar to VACCP-L1R; target of neutralizing antibody; S-S bond formation pathw thiol substrate; myristylprotein (NCBI).
Detailed Gene Information: Click here.

j. Gene Engineering of VACVgp196

Type: Protein
Description:
Detailed Gene Information: Click here.

k. Gene Engineering of VACVgp200

Type: Protein
Description: The cell-associated and released extracellular virions (EV) are thought to be largely responsible for direct cell-to-cell and long-range virus spread within a host, respectively (Fogg et al., 2004).
Detailed Gene Information: Click here.

l. Adjuvant:

VO ID: VO_0001238
Description: Either a Ribi adjuvant system consisting of MPL+TDM or the saponin adjuvant QS-21 was used (Fogg et al., 2004)

m. Adjuvant:

VO ID: VO_0001310

n. Preparation

Soluble forms of L1, A33, and B5 were purified. Recombinant proteins (10 µg) were diluted in PBS with the adjuvant for a total injection volume of 0.1 ml. Monophosphoryl-lipid A plus trehalose dicorynomycolate emulsion (MPL+TDM) was prepared immediately before each immunization according to manufacturer's instructions (Fogg et al., 2004).

o. Virulence

(Fogg et al., 2004)

p. Description

Both intracellular mature virus (IMV) and EV are infectious, but they contain different viral outer membrane proteins, bind to cells differently and have different requirements for entry. Although the entry process is not well understood, a model consistent with available data is that IMV fuse directly with plasma membrane, whereas EV entry involves endocytosis, low-pH-induced disruption of the outer membrane, and fusion of the exposed IMV with the endosomal membrane. Protein subunit vaccines have been evaluated in mice (Fogg et al., 2004).
Recombinant proteins of the outer membranes of IMV and EEV forms of vaccinia virus were used individually or in combination to immunize mice before i.n. challenge with a lethal dose of the WR strain of vaccinia virus. Vaccination with the individual proteins afforded partial protection; complete protection was achieved with 3 doses of the 3-protein IMV–EEV combination vaccine (Parrino et al., 2006).

q. Mouse Response

Host Strain: BALB/c
Vaccination Protocol: Female 5- to 6-week-old BALB/c mice were purchased from Taconic (Germantown, N.Y.). 15 µg of QS-21 aliquots (2 mg/ml in water) were used for each immunization. Proteins were administered at 3-week intervals. Blood was collected from the tail vein 1 day prior to each immunization.
One day prior to challenge, serum samples were collected and mice were weighed. On the day of challenge, an aliquot of purified VV-WR was thawed, sonicated, and diluted in PBS. Mice were anesthetized by inhalation of isoflurane and inoculated i.n. with a 20-µl suspension of 1 x 10^6 or 2 x10^7 PFU of VV-WR. Mice were weighed daily for 2 w following challenge and were euthanatized when they lost 30% of their initial body weight (Fogg et al., 2004).
Persistence: (Fogg et al., 2004)
Side Effects: (Fogg et al., 2004)
Efficacy: Complete survival was obtained with the combination of all three proteins. Although there is a need for safer vaccines, it is difficult to evaluate their efficacy in the absence of human smallpox or information regarding the correlates of immunity (Fogg et al., 2004).
Description: Soluble forms of several vaccinia virus IMV and EV membrane proteins have been engineered to learn more about immunity to poxviruses and to test the proteins as components of a vaccine. The present study involves recombinant L1, A33, and B5 proteins individually or in combinations and then challenged the mice (Fogg et al., 2004).

7. Killed Vaccinia Virus with Adjuvant NanoEmulsion

a. Vaccine Ontology ID:

VO_0004150

b. Type:

Inactivated or "killed" vaccine

c. Antigen

Two strains of vaccinia virus (VV) were used for antigens: VV Western Reserve strain (VVWR) and recombinant Western Reserve strain (VVWR-Luc). The recombinant strain is the same as the VVWR except for expression of firefly luciferase from the pH 7.5 early/late promoter (Bielinska et al., 2008).

d. Adjuvant:

VO ID: VO_0001319

e. Preparation

The viruses killed by incubation were incubated for 3 h at 37°C in 10% NE. Nasal instillation killed virus was diluted to obtain either 1 x 10^3 PFU or 1 x 10^5 PFU per dose in 1% NE. Vaccine formulations containing formalin-killed virus (Fk) were prepared by incubation of VV (about 10^8 PFU/ml) with 0.1% formalin at room temperature for 3 h. This mixture was then diluted in either saline or 1% NE to 1 x 10^3 or 1 x 10^5 PFU per dose to reduce the formalin to nontoxic concentrations for intranasal (i.n.) immunization (Bielinska et al., 2008).

f. Mouse Response

Host Strain: BALB/c
Vaccination Protocol: Mice were vaccinated with 10 to 15 µl of vaccine formulation per naris by use of a pipette tip. Emulsion was applied slowly to minimize bronchial distribution and swallowing of the material (Bielinska et al., 2008).
Immune Response: The intranasal vaccination resulted in both systemic and mucosal anti-VV immunity, virus-neutralizing antibodies, and Th1-biased cellular responses (Bielinska et al., 2008).
Side Effects: Mice protected with VV/NE immunization did have clinical symptoms more extensive than animals vaccinated by scarification post-challenge (Bielinska et al., 2008).
Challenge Protocol: Aliquots of purified recombinant VVWR-Luc or VVWR were thawed and diluted in saline on the day of the challenge. Mice were challenged i.n. with a 20-µl suspension of 2 x 10^6 PFU live VVWR-Luc, corresponding to 10 times the 50% lethal dose, or with live VVWR doses ranging from 1 x 10^7 to 3.2 x 10^3 in fivefold dilutions (Bielinska et al., 2008).
Efficacy: Nasal vaccination with VV/NE vaccine produced protection against lethal infection equal to vaccination by scarification, with 100% survival after challenge (Bielinska et al., 2008).

8. LC16m0

a. Vaccine Ontology ID:

VO_0004098

b. Type:

Live attenuated

c. Preparation

The Lister strain (Elstree) was one of the vaccinia viruses employed in the preparation of vaccines which were widely used in the World Health Organization smallpox eradication program with an acceptable safety record. The LC16 strains were first selected through 36 passages of the original Lister (LO) strain in primary rabbit kidney cells at 30°C. After a further 6 passages, LC16m0 was selected as a temperature-sensitive and medium pock-forming virus, and LC16m8 was cloned from the LC16m0 strain as a small pock-forming variant. The LC16m0 strain was shown to be satisfactorily attenuated in terms of fever response by inoculating nearly 1000 subjects, and the LC16m8 strain was confirmed to be more attenuated, especially in terms of dermal reaction, by tests on more than 100,000 children in Japan in 1974 and 1975, and was licensed as the first attenuated smallpox vaccine (Sugimoto et al., 1994).

d. Virulence

Both LC16m8 and LC16m0 strains are temperature-sensitive and revealed much lower neurovirulence in cynomolgus monkeys and rabbits than the parental virus (LO): viral growth of LC16m0 and LC16m8 in the brain of rabbits and cynomolgus monkeys was at a lower level than that of LO. The genes responsible for neurovirulence and temperature sensitivity do not seem to correlate with the B5R gene and remain to be identified. The identification of these genes will make it possible to improve the VV vectors. The neurovirulence of RVVs tended to be milder than that of the parental vaccinia viruses. The neurovirulence of the WR strain and its recombinant was strongest, followed by that of LO1/LO-RVV (Sugimoto et al., 1994).

e. Description

The LC16m0 strain is one of several temperature-sensitive and further attenuated variants derived from the Lister (Elstree) strain of vaccinia virus, which has a proven safety record in human populations. Several types of recombinant vaccinia viruses expressing a foreign antigen gene from a pathogenic virus have been constructed using the LC16m0 strain as a vector, and their immunological and virological characteristics have been investigated extensively (Sugimoto et al., 1994).

9. LC16m8

a. Vaccine Ontology ID:

VO_0004091

b. Type:

Attenuated Lister strain

c. Gene Engineering of A33R from Vaccinia virus (strain: WR (Western Reserve))

Type: Protein
Description: Similar to VACCP-A33R; associates with A36R; involved in CEV-cell adherence and actin tail formation (NCBI).
Detailed Gene Information: Click here.

d. Gene Engineering of A34R

Type: Protein
Description: Similar to VACCP-A34R; involved in CEV cell adherence and actin tail formation (NCBI).
Detailed Gene Information: Click here.

e. Gene Engineering of A36R

Type: Protein
Description: Similar to VACCP-A36R; interacts with A33R and used in actin tail formation (NCBI).
Detailed Gene Information: Click here.

f. Gene Engineering of A56R

Type: Protein
Description: Similar to VACCP-A56R; EEV; type-I membrane glycoprotein; inhibits cell fusion (NCBI).
Detailed Gene Information: Click here.

g. Gene Engineering of A21L

Type: Protein
Description:
Detailed Gene Information: Click here.

h. Gene Engineering of B5R from Vaccinia virus (strain: WR (Western Reserve))

Type: Protein
Description: Similar to VACCP-B5R; required for trans-Golgi/endosomal membrane-wrapping of IMV (NCBI).
Detailed Gene Information: Click here.

i. Gene Engineering of F13L

Type: Protein
Description: Similar to VACCP-F13L; phospholipase motif, required for IEV formation (NCBI).
Detailed Gene Information: Click here.

j. Preparation

Virus suspensions were diluted serially in phosphate-buffered saline (PBS) supplemented with adjuvant and overlaid with a suspension of 1% carboxyl methyl cellulose in DMEM supplemented to contain 5% FBS (Empig et al., 2006).

k. Virulence

RPV is a virulent strain of vaccinia virus that produces high levels of detectable EEV. In infected rabbits, it causes a generalized, disseminated infection, resulting in death in the majority of cases. However, LC16m8, despite the B5R mutation, is equivalent to Dryvax in the capacity to protect rabbits from lethal RPV challenge (Empig et al., 2006).

l. Description

LC16m8 was developed and widely used in Japan prior to the global eradication of smallpox. Numerous preclinical safety studies in several animal species were conducted comparing LC16m8 to the parental strain Lister, each showing that the attenuated vaccinia virus strain, in contrast to Lister, was incapable of invading the central nervous system (CNS). LC16m8 also was not neuroinvasive in cortisone-treated immunocompromised mice. Clinical trials in Japan, in which over 10,000 children received LC16m8, demonstrated enhanced safety of this attenuated vaccine in comparison to Lister, while confirming that its immunogenicity was unaltered (Empig et al., 2006).

m. Mouse Response

Host Strain: A/NCR
Vaccination Protocol: Three groups of 30 4–6 week old A/NCR mice each were vaccinated at the base of the tail with LC16m8 (approximately 2 × 10^5 PFU). Forty-one days post-vaccination, sera were collected for assessment of virus-specific antibody responses prior to challenge. Forty-nine days post-vaccination, animals were challenged with ECTV delivered by aerosol. ECTV was suspended in DMEM without FBS and inoculated by using a nose-only inhalation exposure system. The remaining mice were observed for 21 days for signs of disease and mortality. Animals were evaluated daily for weight assessment and clinical symptoms (Empig et al., 2006).
Persistence: (Empig et al., 2006)
Side Effects: Despite limiting vaccination to healthy individuals in recent vaccination campaigns, adverse reactions were still observed, highlighting the need for a safer yet equally protective alternative to Dryvax. However, no serious adverse reactions, such as encephalitis, were observed during the early use of LC16m8 vaccine (Empig et al., 2006).
Efficacy: Mice immunized with LC16m8 were protected against lethal ECTV infection. LC16m8 generated antibody responses in mice that exceeded those generated by Dryvax (Empig et al., 2006).
Description: To evaluate the protective efficacy of LC16m8 in comparison to Dryvax, the study employed both rabbit and mouse models of poxvirus disease (Empig et al., 2006).

n. Rabbit Response

Host Strain: New Zeland White (NZW)
Vaccination Protocol: Groups of 20 NZW rabbits (10 male and 10 female) were vaccinated with LC16m8 (at approximately 2 × 10^5 PFU) via scarification to the hind flank. 28 d after vaccination, animals were challenged intradermally with either low (200 PFU) or high (1000 PFU) doses of RPV, which correspond to 1 or 5 times the LD100 value, respectively. Animals were monitored daily for temperature and behavioral changes. Survival was determined at 10 d after RPV challenge, at which time all living rabbits were euthanized (Empig et al., 2006).
Persistence: (Empig et al., 2006)
Side Effects: A mild inflammatory response was detected over the vaccination site 1–7 d following vaccination with either LC16m8 or Dryvax. The response was characterized by ulceration and erythema, followed by pock formation and a small scar. Despite limiting vaccination to healthy individuals in recent vaccination campaigns, adverse reactions were still observed, highlighting the need for a safer yet equally protective alternative to Dryvax. However, no serious adverse reactions, such as encephalitis, were observed during the early use of LC16m8 vaccine (Empig et al., 2006).
Efficacy: Rabbits vaccinated with LC16m8 survived lethal RPV challenge at both challenge doses tested (Empig et al., 2006).
Description: To evaluate the protective efficacy of LC16m8 in comparison to Dryvax, the study employed both rabbit and mouse models of poxvirus disease (Empig et al., 2006).

10. MVA

a. Vaccine Ontology ID:

VO_0004092

b. Type:

Replication-defective virus

c. Preparation

A vial of MVA passage 572 was plaque-purified, propagated in chick embryo fibroblasts, and purified by sedimentation through a sucrose cushion (Earl et al., 2004).

d. Virulence

(Earl et al., 2004; Meseda et al., 2005)

e. Description

Modified vaccinia Ankara (MVA) has been studied most extensively out of the replication-defective vaccines. MVA has an excellent safety profile and could be used in groups in whom Dryvax is currently contraindicated. MVA was given to 120,000 people in Germany in the 1970s, followed by vaccination with live virus Elstree. MVA was safe but was not field-tested because smallpox was not present in Europe at that time. MVA has since been evaluated in animal models and in human studies. In phase I human clinical trials, MVA was found to be safe and immunogenic on its own and found to prime for greater immune responses and attenuate lesion formation if given in advance of Dryvax vaccination. MVA is also being evaluated in persons with contraindications to live virus vaccine such as atopic dermatitis and immunosuppression (Parrino et al., 2006).

f. Monkey Response

Host Strain: Cynomolgous macaque (Macaca fascicularis)
Vaccination Protocol: Monkeys were inoculated with 10^8 plaque-forming units (PFU). 24 monkeys were divided into 4 groups: group 1 received an inoculation with 10^8 PFU of MVA at t = 0 and a second 2 months later; group 2 received one injection with 10^8 PFU of MVA followed 2 months later by a standard percutaneous inoculation with Dryvax; group 3 received nothing at t = 0 and 1 Dryvax inoculation 2 months later; group 4 served as the unimmunized control (Earl et al., 2004).
Persistence: The response to the first MVA inoculation was detected at 1 week, peaked at 2-4 weeks, and was boosted 1 week after the second MVA dose (Earl et al., 2004).
Side Effects: MVA caused no adverse effects, even when high doses were injected into immune-deficient NHPs (Earl et al., 2004).
Efficacy: All immunized animals remained clinically well (Earl et al., 2004).
Description: As vaccines can no longer be tested for their ability to prevent smallpox, licensing will necessarily include comparative immunogenicity and protection studies in non-human primates (NHPs). Here, a highly attenuated MVA is compared with the licensed Dryvax vaccine in an NHP model (Earl et al., 2004).

g. Mouse Response

Host Strain: BALB/cByJ
Vaccination Protocol: To compare the effectiveness of various routes of MVA immunization, male BALB/cByJ mice (obtained from the Jackson Laboratory, Bar Arbor, ME) were immunized through 3 different routes at doses from 10^6 to 10^8 pfu, and sera were collected every 3 weeks for 15 weeks for evaluation of Dryvax-specific antibody by ELISA using inactivated virus (Meseda et al., 2005).
Persistence: The antibody response to vaccination was observed in mice over a relatively long period of time (12–15 weeks) following the initial dose of vaccine. By each measurement, the elicited immune response was stable over this time frame for both Dryvax and MVA. Further, when animals received a second dose of MVA, the antibody response was elevated compared to a single immunization, and was stable for the remainder of the observation period (6 to 9 weeks) (Meseda et al., 2005).
Side Effects: A safer smallpox vaccine could benefit the millions of people that are advised not to take the current one because they or their contacts have increased susceptibility to severe vaccine side effects. Because the correlates of smallpox protection are unknown, findings of similar humoral and cellular immune responses to MVA and Dryvax in NHPs and substantial protection against a severe monkeypox virus challenge are important steps in the evaluation of MVA as a replacement vaccine for those with increased risk of severe side effects from the standard live vaccine, or as a pre-vaccine. As a result of extreme attenuation, MVA causes no adverse effects even when high doses are injected into immunedeficient NHPs. No adverse local or systemic effects were noted after vaccination with MVA. As expected, pustular skin lesions did develop after Dryvax (Earl et al., 2004).
Significant adverse events are associated with vaccination with the currently licensed smallpox vaccine. Candidate new-generation smallpox vaccines, such as MVA, produce very few adverse events in experimental animals and in limited human clinical trials conducted near the end of the smallpox eradication campaign. MVA was administered to more than 120,000 individuals in the latter stages of the smallpox eradication campaign without significant adverse events, although the thoroughness of safety data monitoring at that time is unclear. In addition to a vaccination strategy that employs multiple immunizations of MVA, alternative smallpox vaccination strategies may include an initial vaccination with non-replicating virus vaccine followed by a second immunization with a traditional replicating virus vaccine in order to reduce the possibility of vaccine-associate adverse events due to replicating vaccinia virus. Such a scheme of vaccination may be considered as a means of reducing the rate of adverse events associated with traditional smallpox vaccination, provided that vaccine efficacy is not compromised (Meseda et al., 2005).
Efficacy: Mice immunized intradermally (i.d.) with either 10^8 pfu of MVA, or a prime-boost combination of 10^8 pfu of MVA followed by either 10^6 pfu of Dryvax or 10^8 pfu of MVA survived an intranasal (i.n.) challenge with 25 LD50s of vaccinia virus WR. Furthermore, vaccination with a single dose of 10^8 pfu of MVA resulted in a minimal weight loss (<10%), as did a vaccination combination of 10^8 pfu of MVA followed by 10^6 pfu of Dryvax. When mice that were immunized with a lower dose of 10^6 pfu of MVA were challenged with i.n. vaccinia virus WR, 4/5 survived a challenge with 10 LD50s at either 6 weeks or 12 weeks post-vaccination. When mice received a single immunization of 10^6 pfu of MVA and were challenged with 25 LD50s, 4/5 survived challenge at 6 weeks post-vaccination and 3/5 survived challenge at 12 weeks post-vaccination. In contrast, all animals receiving 10^6 MVA and boosted 6 weeks later with either 10^6 pfu of MVA or 10^6 pfu of Dryvax survived. These results indicate that combinations of MVA are as effective as Dryvax in eliciting immune responses and inducing protective immunity in a mouse model (Meseda et al., 2005).
Description: The aim of the present study was to compare the immunogenicity and protective ability of MVA (a leading candidate new-generation smallpox vaccine) to the licensed smallpox vaccine Dryvax in a mouse model of vaccination. MVA is a replication-defective vaccinia virus derived from the Ankara strain by more than 500 passages through primary chicken embryo fibroblasts (CEF). This virus grows to high titer in CEF cells but replicates poorly, if at all, in human cells (Meseda et al., 2005).

11. MVA-BN

a. Vaccine Ontology ID:

VO_0004097

b. Type:

Highly attenuated clone

c. Preparation

MVA-BN has been derived via additional passages in serum free chicken embryo fibroblast (CEF) cultures, and is replication incompetent in mammalian cell lines, avirulent even in immune compromised hosts, highly immunogenic in mammalian animal models, and may be administered both s.c. and i.m. The vaccine was produced by IDT under Good Manufacturing Practice (GMP) conditions and provided by Bavarian Nordic as a liquid frozen product stored at −80 °C. Doses of 2 × 10^6, 2 × 10^7, 2 × 10^8 TCID50/ml were formulated in 10 mM Tris, 140 mM NaCl, pH 7.4. The vaccine was thawed and 0.5 ml were administered to subjects to deliver a dose of 10^6, 10^7, 10^8 TCID50, respectively (Vollmar et al., 2006).

d. Virulence

(Vollmar et al., 2006)

e. Description

MVA-BN (IMVAMUNE) was developed from the Modified Vaccinia Ankara strain (MVA) that was used as a priming vaccine prior to administration of conventional smallpox vaccine in a two-step program and shown to be safe in more than 120,000 primary vaccinees in Germany and used as a veterinary vaccine to protect against several veterinary orthopoxvirus infections (Vollmar et al., 2006).

f. Human Response

Host Strain: Adult males
Vaccination Protocol: Healthy male subjects aged 20–55 years were eligible for recruitment. The study design was divided into two parts: Part I subjects (n = 68) had no prior history of smallpox vaccination, while Part II (n = 18) subjects had a prior history of smallpox vaccination, documented by a vaccination certificate or a typical vaccination scar. Part I subjects were randomly assigned to receive a dose of either 10^6 (Group 1, n = 18), 10^7 (Group 2, n = 16), 10^8 (Group 3, n = 16) TCID50 MVA-BN in a double-blind manner, or 10^8 TCID50 (Group 4, n = 18) open-label, on day 0 and d28. Part II participants received a single dose of 10^8 TCID50 (Group 5, n = 18), open-label, to evaluate a boost response in previously vaccinated subjects. Study-specific assessments were conducted at screening and on d 0, 7, 28, 35, 42, and 126 (Vollmar et al., 2006).
Persistence: T-cell immunity can persist for up to 50 years after immunization (Vollmar et al., 2006).
Side Effects: 15 of the 64 general adverse events were assessed as possibly related to the study vaccine. 2 of these each occurred in Groups 1, 3, and 4, respectively, and 9 (28%) in the pre-immunized subjects. Only 1 serious adverse event was reported during the study: a subject in Group 4 was hospitalized due to an infected epidermal cyst on the face, 12 days after the second injection; however, this event was judged to be unrelated to the study vaccine, and the subject recovered without sequelae (Vollmar et al., 2006).
Efficacy: The immune responses achieved after administration of MVA-BN were highly dose-dependent. Total IgG seropositivity rates, as determined by the ELISA, reached a maximum of 81% and 88% following a single vaccination using the highest dose (10^8 TCID50) via the s.c. or i.m. routes, respectively. They reached 100% following the second vaccination. On the other hand, the pre-immunized subjects attained 100% seropositivity after a single vaccination with MVA-BN although only 4 of these subjects had detectable antibody titers prior to inclusion, implying a pre-existing boostable immunity more than 20 years post-vaccination (Vollmar et al., 2006).
Description: The primary objective of the study was to demonstrate safety and tolerability of MVA-BN at different doses administered to healthy subjects with or without a history of smallpox vaccination. Immunogenicity was assessed in all subjects as a secondary endpoint and was also used to evaluate dose-related responses and optimal route of application (Vollmar et al., 2006).

g. Monkey Response

Host Strain: cynomolgus macaques (Macaca fascicularis)
Vaccination Protocol: Four groups of six captive-bred sub-adult healthy monkeys each were vaccinated: the first group was vaccinated twice with a high dose of 10^8 TCID50 MVA-BN at an interval of 4 weeks, the second group was vaccinated with a low dose of 2 x 10^6 TCID50 MVA-BN followed 10 days later by a s.c. vaccination with Elstree-RIVM, the third group was vaccinated with one s.c. standard dose of Elstree-RIVM, and the fourth group was vaccinated s.c. with one standard dose of Elstree-BN. Group V was sham vaccinated. 15 weeks after the last vaccination, all of the animals were challenged intratracheal (i.t.) with either 10^6 PFU (3 animals per group) or 10^7 PFU (3 animals per group) of MPXV, which were chosen as sub-lethal and lethal challenges, respectively (Stittelaar et al., 2005).
Persistence: (Stittelaar et al., 2005)
Side Effects: Elevated body temperatures were observed (Stittelaar et al., 2005).
Efficacy: All vaccinated animals that were challenged showed an episode of elevated body temperature (>1°C; ~2.65%) that occurred between days 5 and 8 post-challenge which returned to normal by d 12. Only one vaccinated animal developed pocks upon MPXV challenge, while all others showed no clinical signs of the disease apart from an elevated body temperature. This animal, which was vaccinated with MVA-BN (group I), initially developed pocks (>70) on d 11 after the challenge with MPXV (Stittelaar et al., 2005).
Description: The present study investigated different combinations of candidate and traditional vaccines, followed by MPXV challenge i.t. The MVA strain (MVA-BN, or IMVAMUNE) is currently being tested in >300 human subjects in on-going phase I and II clinical studies, including individuals for whom vaccination with traditional smallpox vaccines is traditionally contraindicated. For the present study, the immune response and efficacy of MVA-BN vaccination were compared to those of a primary vaccination with MVA-BN followed by vaccination with a first-generation smallpox vaccine produced on calf skins (Elstree-RIVM). For this purpose, a low dose of MVA was chosen to prime the immune system, thus reducing the side effects of vaccination with a traditional vaccine shortly thereafter without changing the take rate of the traditional vaccine. In addition, vaccination protocols with Elstree-RIVM alone and vaccination with a second-generation vaccine (Elstree-BN) were evaluated. Elstree-BN is based on the same vaccinia virus strain as Elstree-RIVM, but the former was passaged and produced on chicken embryo fibroblasts to further attenuate the virus and to make a better defined vaccine preparation that does not depend on the use of calves (Stittelaar et al., 2005).

12. NYVAC

a. Vaccine Ontology ID:

VO_0004093

b. Type:

Replication defective virus

c. Preparation

NYVAC was derived from the Copenhagen strain and developed by selective deletion of 18 open reading frames (ORFs) (Parrino et al., 2006).

d. Virulence

(Belyakov et al., 2003; Edghill-Smith et al., 2003)

e. Description

Smallpox vaccination induced significantly larger skin lesions in immunocompromised macaques than in healthy macaques. Vaccination of immunocompromised macaques with the genetically-engineered, replication-deficient poxvirus NYVAC, before or after retrovirus infection, was safe and lessened the severity of Dryvax-induced skin lesions. Neutralizing antibodies to vaccinia were induced by NYVAC, even in macaques with severe CD4+ T cell depletion, and their titers inversely correlated with the time to complete resolution of the skin lesions. Together, these results provide the proof of concept, in macaque models that mirror human immunodeficiency virus (HIV) type 1 infection, that a prime-boost approach with a highly attenuated poxvirus followed by Dryvax increases the safety of smallpox vaccination, and they highlight the importance of neutralizing antibodies in protection against virulent poxvirus (Edghill-Smith et al., 2003).

f. Mouse Response

Host Strain: BALB/c, B cell-deficient, and CD1 KO–/– mice
Vaccination Protocol: Female BALB/c mice (6–10 weeks old, purchased from Frederick Cancer Research Facility, Frederick, MD), B cell-deficient (Taconic Farms), and CD1 KO-/- (CD1KO, from M. Grusby) mice were innoculated with NYVAC at doses from 10^3 to 10^7 pfu. For comparison, and as a positive control, immunization with Wyeth human vaccine strain of vaccinia virus was given by tail scratch (corresponding to skin scratch used for human vaccination). One month after immunization, mice were challenged with 10^6 pfu of WR by intranasal (i.n.) inoculation (Belyakov et al., 2003).
Persistence: (Belyakov et al., 2003)
Side Effects: None were mentioned. Replication-defective strains might be valuable as a preliminary immunization to reduce the risk of serious adverse ffects of conventional smallpox vaccination (Belyakov et al., 2003).
Efficacy: Protection at most doses of NYVAC given i.m. was roughly comparable to that produced by the corresponding doses of MVA given i.m., and no statistically significant difference was detected. It was found that i.m. injection with MVA induced protection of immunized animals in a dose-dependent manner. A dose of 10^7 pfu of MVA given i.m. induced complete protection against challenge with WR (Belyakov et al., 2003).
Description: At sufficient doses, the protection provided by modified NYVAC replication-deficient vaccinia viruses, safe in immunocompromised animals, was equivalent to that of the licensed Wyeth vaccine strain against a pathogenic vaccinia virus i.n. challenge of mice. A similar variety and pattern of immune responses were involved in protection induced by modified vaccinia Ankara and Wyeth viruses. For both, antibody was essential to protect against disease, whereas neither effector CD4+ nor CD8+ T cells were necessary or sufficient. However, in the absence of antibody, T cells were necessary and sufficient for survival and recovery. Also, T cells played a greater role in control of sublethal infection in unimmunized animals. These properties, shared with the existing smallpox vaccine, provide a basis for further evaluation of these replication-deficient vaccinia viruses as safer vaccines against smallpox or against complications from vaccinia virus (Belyakov et al., 2003).

g. Monkey Response

Host Strain: Indian rhesus macaques
Vaccination Protocol: Twenty-five monkeys were enrolled: 6 of the macaques were immunocompetent (groups 1 and 2), and macaques in group 1 were vaccinia naive. Macaques in group 2 had been exposed previously to the attenuated nef- SIVmac239 strain. They were immunized with a single inoculation of NYVAC 1 month before Dryvax vaccination. Group 3 included 7 macaques, 3 that had been infected with the chimeric SHIV 89.6 PD strain for 8 months, 3 that had been infected with the SIVmac251 strain for 12 months, and 1 that had been infected with the nef- SIVmac239 strain for 32 months. Four macaques (group 5) that, at first, had been infected with the same SIVmac251 strain and that subsequently were vaccinated with 3 inoculations of NYVAC, at weeks 10, 19, and 23 after infection (for macaques 480, 644, and H684) or at weeks 42, 48, and 54 after infection (for macaque 3143), were used. The overall time of SIVmac251 infection was 41 months for macaques 480 and 644 and 25 months for macaques H684 and 3143. Four macaques had been infected with SHIV 89.6 PD for 12 months. They were vaccinated with 3 inoculations of NYVAC (10^8 pfu) 6 weeks apart and were challenged with Dryvax 6 months after the final NYVAC immunization. All 25 macaques were vaccinated with Dryvax at the same dose at the times indicated. In brief, the bifurcated needle was immersed in the vaccine suspension and was used to poke the skin 15 consecutive times, in accordance with US Food and Drug Administration (FDA) guidelines. The lesions that developed after smallpox vaccination were photographed every 2 days and were imaged by manually defining the topographic contours of the affected skin (Edghill-Smith et al., 2003).
Persistence: The immunocompromised macaques were vaccinated with NYVAC at 6 months to a maximum of 36 months before Dryvax challenge, suggesting that this vaccine is able to induce lasting immune responses even as CD4+ helper T cells are progressively depleted. However, the lag between NYVAC and Dryvax vaccinations appears to be important (Edghill-Smith et al., 2003).
Side Effects: The prime-boost approach with a highly attenuated poxvirus followed by Dryvax increases the safety of smallpox vaccination (Edghill-Smith et al., 2003).
Efficacy: The prime-boost approach with a highly attenuated poxvirus followed by Dryvax increases the safety of smallpox vaccination, and highlights the importance of neutralizing antibodies in protection against virulent poxvirus (Edghill-Smith et al., 2003).
Description: The replication competence of live vaccines, such as the only currently available smallpox vaccine, Dryvax, may pose safety concerns when injected in individuals with congenital, acquired, or iatrogenic immunodeficiency. Because the number of patients with immunodeficiency has increased worldwide as a result of the HIV-1 epidemic, the increase in the number of organ transplants, and aggressive chemotherapy in patients with cancer, the risks associated with Dryvax vaccination may affect a larger portion of the population than before. It has been hypothesized that immunization of immunocompromised individuals, with highly attenuated poxviruses, may ameliorate the clinical outcome of Dryvax vaccination. In macaques with modest to severe depletion of CD4+ T cells, it was tested whether immunization with NYVAC before or after infection with simian immunodeficiency virus (SIV) or simian/human immunodeficiency virus (SHIV) would increase the safety of Dryvax vaccination. NYVAC was shown to be safer in severely immunocompromised macaques and that NYVAC priming resulted in a faster resolution of Dryvax-induced lesions in both healthy and immunocompromised macaques (Edghill-Smith et al., 2003).

13. Recombinant vaccinia A27L, D8L, and B5R Proteins with adjuvant MPL-TDM

a. Vaccine Ontology ID:

VO_0004149

b. Type:

Subunit vaccine

c. Antigen

This vaccine uses the A27L and D8L proteins from the intracellular mature virus form and the B5R protein from the extracellular enveloped virus form of the vaccinia virus (Berhanu et al., 2008).

d. Adjuvant:

VO ID: VO_0001250
Description: The adjuvant used in this vaccine was Monophosphoryl Lipid A and Trehalose Dicorynomycolate (MPL-TDM) (Berhanu et al., 2008).

e. Preparation

Vaccines contained 10 micrograms of A27L, D8L, and B5R in 100 microliters of phosphate buffered saline mixed with 100 microliters of MPL-TDM (Berhanu et al., 2008).

f. Mouse Response

Host Strain: BALB/c
Vaccination Protocol: Six-week old mice were immunized three times subcutaneously at weeks 0, 3, and 5 (Berhanu et al., 2008).
Immune Response: Three immunization with the proteins alone induced potent neutralizing antibody reponses (Berhanu et al., 2008).
Challenge Protocol: Five weeks or two weeks after the last immunization, mice were challenged intranasally with with 20 LD50 VV-WR in 20 microliters of PBS by applying equally between the two nares (Berhanu et al., 2008).
Efficacy: Immunization provided complete protection against lethal viral challenge. Several linear B-cell epitopes within the three proteins were recognized by serum from the immunized mice. In addition protein-specific cellular responses were detected in spleens of immunized mice by gamma interferon (Berhanu et al., 2008).

14. Smallpox DNA Vaccine

a. Vaccine Ontology ID:

VO_0004096

b. Type:

DNA

c. Gene Engineering of A27L

Type: Protein
Description:
Detailed Gene Information: Click here.

d. Gene Engineering of A33R from Monkeypox virus (strain: Zaire-96-I-16)

Type: Protein
Description:
Detailed Gene Information: Click here.

e. Gene Engineering of B5R from Monkeypox virus (strain: Zaire-96-I-16)

Type: Protein
Description: (Hooper et al., 2004)
Detailed Gene Information: Click here.

f. Gene Engineering of L1R from Monkeypox virus Zaire-96-I-16

Type: Protein
Description:
Detailed Gene Information: Click here.

g. Preparation

The 4pox DNA vaccine contained two IMV-specific genes (L1R and A27L) and two EEV-specific genes (A33R and B5R) (Hooper et al., 2004).

h. Virulence

(Hooper et al., 2004)

i. Description

DNA vaccine strategies have been investigated in animal models. A DNA vaccine composed of 4 vaccinia virus genes protected rhesus macaques from severe disease, with the animals exhibiting mild clinical and laboratory abnormalities, after challenge with a lethal dose of monkeypox virus. When vaccinated with a single gene (L1R), macaques developed severe, but not fatal, disease. Heterologous prime-boost strategies have also been evaluated. Priming BALB/c mice with DNA vaccine resulted in greater immune responses after boosting with live vaccinia virus compared with controls (Parrino et al., 2006).

j. Human Response

Host Strain: rhesus macaque (Macaca mulata)
Vaccination Protocol: The challenge experiment included 4 groups: group 1 consisted of 3 monkeys vaccinated with the 4pox DNA vaccine, group 2 consisted of 2 monkeys vaccinated with the L1R DNA vaccine, group 3 (negative controls) consisted of 3 monkeys vaccinated with a Hantaan virus DNA vaccine, and group 4 (positive controls) consisted of 2 monkeys vaccinated with the human smallpox vaccine (Dryvax). The L1R DNA vaccine was tested to determine the degree to which vaccination with a single immunogen eliciting IMV-neutralizing antibodies could confer protection. The DNA vaccines were administered by gene gun. Five weeks before challenge, all monkeys, except the monkeys vaccinated with Dryvax and one of the negative controls, were vaccinated with new preparations of the same DNA vaccine they had received 1-2 years earlier. This booster vaccination was administered to affirm that immunological memory had been elicited by the initial vaccination series and to ensure robust responses to the DNA vaccines with the intent to prove concepts rather than explore minimal requirements for protection. Based on the dosing experiments, a dose of 2 x 10^7 PFU was chosen for the vaccine evaluation experiment. Vaccinated monkeys were challenged with MPOV-Z79 by i.v. injection into the right or left saphenous vein. At 2-day intervals, whole-blood, serum, and throat swab samples were collected, and rectal temperature, pulse, and blood oxygen saturation were measured (Hooper et al., 2004).
Persistence: Gene gun vaccination with the 4pox DNA vaccine or the L1R DNA vaccine elicited a memory response that was maintained for at least a year and up to 2 years (Hooper et al., 2004).
Side Effects: Although VACV is highly immunogenic and is known to confer long-lasting protective immunity to smallpox, the adverse events associated with the present smallpox vaccine (i.e., Dryvax) pose a significant obstacle to successful vaccination campaigns. Adverse events historically associated
with VACV range from the nonserious (e.g., fever, rash, headache, pain, and fatigue) to life threatening (e.g., eczema vaccinatum, encephalitis, and progressive vaccinia). Serious adverse events that are not necessarily causally associated with vaccination, including myocarditis and/or myopericarditis, have been reported during past and present smallpox vaccination programs. Several adverse cardiac events reported in the first 4 months of the 2003 civilian and military vaccination campaigns prompted the CDC to revise their recommendations for exclusion of potential smallpox recipients to include those persons with heart disease or several other conditions. In addition, identifying protective immunogens might allow the development of a subunit smallpox vaccine that affords protection with negligible adverse events (Hooper et al., 2004).
Efficacy: Monkeys vaccinated with the 4pox DNA vaccine were protected not only from lethal monkeypox but also from severe disease. This is the first demonstration that vaccination with a combination of VACV immunogens, rather than the whole infectious virus, is sufficient to protect NHPs against any poxvirus disease (Hooper et al., 2004).
Description: Much of the threat posed by orthopoxviruses could be eliminated by vaccination; however, because the smallpox vaccine is a live orthopoxvirus vaccine administered to the skin, the vaccine itself can pose a serious health risk. The present study demonstrates that monkeys vaccinated with a DNA vaccine consisting of four vaccinia virus genes (L1R, A27L, A33R, and B5R) were protected from severe disease after an otherwise lethal challenge with monkeypox virus. Animals vaccinated with a single gene (L1R), which encodes a target of neutralizing antibodies, developed severe disease but survived. This is the first demonstration that a subunit vaccine approach to smallpox-monkeypox immunization is feasible (Hooper et al., 2004).

k. Mouse Response

Host Strain: BALB/c
Vaccination Protocol: Adult (16–23 g) female BALB/c mice were vaccinated in the skin of the thigh using an Easy Vax™ DNA vaccine delivery system to deliver the vaccine plasmids on weeks 0, 3, and 8. Anesthetized mice were scarified by placing 10 μl of PBS containing live VACV on the tail. A 26 gauge 5/8" needle was used to scratch the tail to facilitate infection/vaccination. A lesion (pock) at the site of scarification on d 10 indicated successful vaccination. Mice were anesthetized and weighed before i.n. injection of 50 μl of PBS containing 2 × 10^6 pfu of VACV strain IHD-J using a plastic pipette tip. After challenge, mice were observed and weighed daily for 3 weeks (Hooper et al., 2006).
Persistence: (Hooper et al., 2006)
Side Effects: There are several drawbacks to the current anthrax vaccines including nonserious and serious adverse events that make the vaccine contraindicated in large segments of the population (e.g., persons who are immunodeficient, immunosuppressed, pregnant, breastfeeding, or have history of cardiac disease), and because this vaccine results in a localized skin infection containing infectious virus (i.e. pock), the infection can spread to other sites on the body (e.g. ocular autoinoculation) or to persons who come in close contact with the vaccinee. Identification of the genes associated with protective immunity and, conversely, the genes associated with adverse events unrelated to dissemination or transmission will be important for characterizing the next-generation smallpox vaccines and for engineering future smallpox vaccines (Hooper et al., 2006).
Efficacy: Mice vaccinated with the 4pox DNA vaccine using the Easy Vax™ device were completely protected from i.n. challenge with >10 LD50 of VACV, strain IHD-J (Hooper et al., 2006).
Description: The enhanced immunogenicity of DNA vaccines delivered by gene gun likely involves the direct introduction of plasmid DNA to cells in the skin, including specialized antigen-presenting cells (APCs). While the gene gun has yielded among the most promising immune responses for a DNA vaccine thus far, there is the possibly that all of the criteria required for successful product development will not be satisfied. Hence, it is important to continue to evaluate alternative technologies that might better facilitate the development of licensed human vaccines. Alternative means of delivering DNA vaccines under investigation include the use of electric field technologies. Electroporation is a process whereby cells are transiently permeabilized by high-intensity electric field pulses. The present study tests a novel device capable of targeting electroporation to the dermis using a microneedle array. The plasmid DNA is dried onto the tips of the microneedles, which are inserted into the skin where the DNA dissolves in interstial fluid and is then transfected into the surrounding cells by electroporation (Hooper et al., 2006).

IV. References

1. Amanna et al., 2006: Amanna IJ, Slifka MK, Crotty S. Immunity and immunological memory following smallpox vaccination. Immunological reviews. 2006 Jun; 211; 320-37. [PubMed: 16824139 ].

2. Belyakov et al., 2003: Belyakov IM, Earl P, Dzutsev A, Kuznetsov VA, Lemon M, Wyatt LS, Snyder JT, Ahlers JD, Franchini G, Moss B, Berzofsky JA. Shared modes of protection against poxvirus infection by attenuated and conventional smallpox vaccine viruses. Proceedings of the National Academy of Sciences of the United States of America. 2003 Aug 5; 100(16); 9458-63. [PubMed: 12869693 ].

3. Berhanu et al., 2008: Berhanu A, Wilson RL, Kirkwood-Watts DL, King DS, Warren TK, Lund SA, Brown LL, Krupkin AK, Vandermay E, Weimers W, Honeychurch KM, Grosenbach DW, Jones KF, Hruby DE. Vaccination of BALB/c mice with Escherichia coli-expressed vaccinia virus proteins A27L, B5R, and D8L protects mice from lethal vaccinia virus challenge. Journal of virology. 2008; 82(7); 3517-3529. [PubMed: 18199639].

4. Bielinska et al., 2008: Bielinska AU, Chepurnov AA, Landers JJ, Janczak KW, Chepurnova TS, Luker GD, Baker JR Jr. A novel, killed-virus nasal vaccinia virus vaccine. Clinical and vaccine immunology : CVI. 2008; 15(2); 348-358. [PubMed: 18057181].

5. Casey et al., 2005: Casey CG, Iskander JK, Roper MH, Mast EE, Wen XJ, Torok TJ, Chapman LE, Swerdlow DL, Morgan J, Heffelfinger JD, Vitek C, Reef SE, Hasbrouck LM, Damon I, Neff L, Vellozzi C, McCauley M, Strikas RA, Mootrey G. Adverse events associated with smallpox vaccination in the United States, January-October 2003. JAMA : the journal of the American Medical Association. 2005 Dec 7; 294(21); 2734-43. [PubMed: 16333009 ].

6. Davies et al., 2005: Davies DH, McCausland MM, Valdez C, Huynh D, Hernandez JE, Mu Y, Hirst S, Villarreal L, Felgner PL, Crotty S. Vaccinia virus H3L envelope protein is a major target of neutralizing antibodies in humans and elicits protection against lethal challenge in mice. Journal of virology. 2005; 79(18); 11724-11733. [PubMed: 16140750].

7. Earl et al., 2004: Earl PL, Americo JL, Wyatt LS, Eller LA, Whitbeck JC, Cohen GH, Eisenberg RJ, Hartmann CJ, Jackson DL, Kulesh DA, Martinez MJ, Miller DM, Mucker EM, Shamblin JD, Zwiers SH, Huggins JW, Jahrling PB, Moss B. Immunogenicity of a highly attenuated MVA smallpox vaccine and protection against monkeypox. Nature. 2004 Mar 11; 428(6979); 182-5. [PubMed: 15014500].

8. Edghill-Smith et al., 2003: Edghill-Smith Y, Venzon D. Modeling a safer smallpox vaccination regimen, for human immunodeficiency virus type 1-infected patients, in immunocompromised macaques. The Journal of infectious diseases. 2003 Oct 15; 188(8); 1181-91. [PubMed: 14551889 ].

9. Empig et al., 2006: Empig C, Kenner JR, Perret-Gentil M, Youree BE, Bell E, Chen A, Gurwith M, Higgins K, Lock M, Rice AD, Schriewer J, Sinangil F, White E, Buller RM, Dermody TS, Isaacs SN, Moyer RW. Highly attenuated smallpox vaccine protects rabbits and mice against pathogenic orthopoxvirus challenge. Vaccine. 2006 Apr 24; 24(17); 3686-94. [PubMed: 16430997 ].

10. FDA: ACAM2000: FDA: ACAM2000 Vaccine for Variola Virus [http://www.fda.gov/BiologicsBloodVaccines/Vaccines/ApprovedProducts/ucm094065.htm]

11. FDA: Dryvax: FDA: Dryvax Vaccine for Variola Virus [http://www.fda.gov/BiologicsBloodVaccines/Vaccines/ApprovedProducts/ucm094066.htm]

12. Fogg et al., 2004: Fogg C, Lustig S, Whitbeck JC, Eisenberg RJ, Cohen GH, Moss B. Protective immunity to vaccinia virus induced by vaccination with multiple recombinant outer membrane proteins of intracellular and extracellular virions. Journal of virology. 2004 Oct; 78(19); 10230-7. [PubMed: 15367588 ].

13. Franz et al., 1997: Franz DR, Jahrling PB, Friedlander AM, McClain DJ, Hoover DL, Bryne WR, Pavlin JA, Christopher GW, Eitzen EM Jr. Clinical recognition and management of patients exposed to biological warfare agents. JAMA : the journal of the American Medical Association. 1997 Aug 6; 278(5); 399-411. [PubMed: 9244332].

14. Golden et al., 2008: Golden JW, Josleyn MD, Hooper JW. Targeting the vaccinia virus L1 protein to the cell surface enhances production of neutralizing antibodies. Vaccine. 2008; 26(27-28); 3507-3515. [PubMed: 18485547].

15. Grabenstein et al., 2003: Grabenstein JD, Winkenwerder W Jr. US military smallpox vaccination program experience. JAMA : the journal of the American Medical Association. 2003 Jun 25; 289(24); 3278-82. [PubMed: 12824209 ].

16. Greenberg et al., 2005: Greenberg RN, Kennedy JS, Clanton DJ, Plummer EA, Hague L, Cruz J, Ennis FA, Blackwelder WC, Hopkins RJ. Safety and immunogenicity of new cell-cultured smallpox vaccine compared with calf-lymph derived vaccine: a blind, single-centre, randomised controlled trial. Lancet. 2005 Jan 29-Feb 4; 365(9457); 398-409. [PubMed: 15680454 ].

17. Hassett, 2003: Hassett DE. Smallpox infections during pregnancy, lessons on pathogenesis from nonpregnant animal models of infection. Journal of reproductive immunology. 2003 Oct; 60(1); 13-24. [PubMed: 14568674].

18. Henderson, 1999: Henderson DA. Smallpox: clinical and epidemiologic features. Emerging infectious diseases. 1999 Jul-Aug; 5(4); 537-9. [PubMed: 10458961].

19. Hooper et al., 2004: Hooper JW, Thompson E, Wilhelmsen C, Zimmerman M, Ichou MA, Steffen SE, Schmaljohn CS, Schmaljohn AL, Jahrling PB. Smallpox DNA vaccine protects nonhuman primates against lethal monkeypox. Journal of virology. 2004 May; 78(9); 4433-43. [PubMed: 15078924].

20. Hooper et al., 2006: Hooper JW, Golden JW, Ferro AM, King AD. Smallpox DNA vaccine delivered by novel skin electroporation device protects mice against intranasal poxvirus challenge. Vaccine. 2006 Nov 27; ; . [PubMed: 17240007].

21. Maksiutov and Shchelkunov, 2011: Maksiutov RA, Shchelkunov SN. [Comparison of protective properties of the smallpox DNA-vaccine based on the variola virus A30L gene and its variant with modified codon usage]. Molekuliarnaia genetika, mikrobiologiia i virusologiia. 2011; (2); 30-34. [PubMed: 21786633].

22. McFadden, 2005: McFadden G. Poxvirus tropism. Nature reviews. Microbiology. 2005 Mar; 3(3); 201-13. [PubMed: 15738948].

23. Meseda et al., 2005: Meseda CA, Garcia AD, Kumar A, Mayer AE, Manischewitz J, King LR, Golding H, Merchlinsky M, Weir JP. Enhanced immunogenicity and protective effect conferred by vaccination with combinations of modified vaccinia virus Ankara and licensed smallpox vaccine Dryvax in a mouse model. Virology. 2005 Sep 1; 339(2); 164-75. [PubMed: 15993917 ].

24. Monath et al., 2004: Monath TP, Caldwell JR, Mundt W, Fusco J, Johnson CS, Buller M, Liu J, Gardner B, Downing G, Blum PS, Kemp T, Nichols R, Weltzin R. ACAM2000 clonal Vero cell culture vaccinia virus (New York City Board of Health strain)--a second-generation smallpox vaccine for biological defense. International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases. 2004 Oct; 8 Suppl 2; S31-44. [PubMed: 15491873].

25. Morikawa et al., 2005: Morikawa S, Sakiyama T, Hasegawa H, Saijo M, Maeda A, Kurane I, Maeno G, Kimura J, Hirama C, Yoshida T, Asahi-Ozaki Y, Sata T, Kurata T, Kojima A. An attenuated LC16m8 smallpox vaccine: analysis of full-genome sequence and induction of immune protection. Journal of virology. 2005 Sep; 79(18); 11873-91. [PubMed: 16140764].

26. NCBI: Entrez Gene [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=gene]

27. Ober et al., 2002: Ober BT, Bruhl P, Schmidt M, Wieser V, Gritschenberger W, Coulibaly S, Savidis-Dacho H, Gerencer M, Falkner FG. Immunogenicity and safety of defective vaccinia virus lister: comparison with modified vaccinia virus Ankara. Journal of virology. 2002 Aug; 76(15); 7713-23. [PubMed: 12097585].

28. Parrino et al., 2006: Parrino J, Graham BS. Smallpox vaccines: Past, present, and future. The Journal of allergy and clinical immunology. 2006 Dec; 118(6); 1320-6. [PubMed: 17157663 ].

29. PathPort: Virginia Bioinformatics Institute [http://pathport.vbi.vt.edu/pathinfo/pathogens/Clostridium_botulinum_Info.shtml]

30. Slifka, 2004: Slifka MK. Immunological memory to viral infection. Current opinion in immunology. 2004 Aug; 16(4); 443-50. [PubMed: 15245737].

31. Stittelaar et al., 2005: Stittelaar KJ, van Amerongen G, Kondova I, Kuiken T, van Lavieren RF, Pistoor FH, Niesters HG, van Doornum G, van der Zeijst BA, Mateo L, Chaplin PJ, Osterhaus AD. Modified vaccinia virus Ankara protects macaques against respiratory challenge with monkeypox virus. Journal of virology. 2005 Jun; 79(12); 7845-51. [PubMed: 15919938 ].

32. Sugimoto et al., 1994: Sugimoto M, Yamanouchi K. Characteristics of an attenuated vaccinia virus strain, LC16m0, and its recombinant virus vaccines. Vaccine. 1994 Jun; 12(8); 675-81. [PubMed: 8091843].

33. Vilesova et al., 1985: Vilesova IS, Gurvich EB, Dzagurov SG, Grigor'eva LV, Abel H. [Changes in the properties of the vaccinia virus isolated in postvaccinal encephalitis]. Voprosy virusologii. 1985 Jul-Aug; 30(4); 477-82. [PubMed: 2865855].

34. Vollmar et al., 2006: Vollmar J, Arndtz N, Eckl KM, Thomsen T, Petzold B, Mateo L, Schlereth B, Handley A, King L, Hulsemann V, Tzatzaris M, Merkl K, Wulff N, Chaplin P. Safety and immunogenicity of IMVAMUNE, a promising candidate as a third generation smallpox vaccine. Vaccine. 2006 Mar 15; 24(12); 2065-70. [PubMed: 16337719].

35. Weltzin et al., 2003: Weltzin R, Liu J, Pugachev KV, Myers GA, Coughlin B, Blum PS, Nichols R, Johnson C, Cruz J, Kennedy JS, Ennis FA, Monath TP. Clonal vaccinia virus grown in cell culture as a new smallpox vaccine. Nature medicine. 2003 Sep; 9(9); 1125-30. [PubMed: 12925845 ].