Supplemental Table 1A: VACV-derived ORFs containing CD8+ T cell epitopes

ORF # VACWR# (Name) GenBank accession # Size 9- and 10-mers Description 12 K ORF B 79 141 20 VACWR062(E6R) 66275859 567 1117 unknown 21 VACWR063(E7R) 66275860 166 315 soluble, myristylprotein has N-myristyltransferase target MGxxxS/T/A 25 VACWR077(I8R) 66275874 676 1335 RNA-helicase, DExH-NPH-II; DNA-helicase aided by L4R 26 VACWR080(G2R) 66275877 220 423 late transcription elongation factor 27 VACWR084(G6R) 66275881 165 313 unknown 29 VACWR086(G8R) 66275883 260 503 late gene transcription VLTF-1 30 VACWR087(G9R) 66275884 340 663 myristylprotein 31 VACWR091(L4R) 66275888 251 485 core protein vp8; microtubule-associated, ssRNA/ssDNA-binding protein 34 VACWR094(J2R) 66275891 177 337 thymidine 35 VACWR095(J3R) 66275892 333 649 multifunctional poly-A subunit, cap methyltransferase, and transcription elongation factor 36 VACWR096(J4R) 66275893 185 353 DNA-dependent RNA polymerase subunit rpo22 37 VACWR098(J6R) 66275895 1287 2557 DNA-dependent RNA polymerase subunit rpo147 42 VACWR106(D1R) 66275903 844 1671 large subunit of mRNA capping ; transcription termination factor VTF 45 VACWR109(D4R) 66275906 218 419 uracil-DNA glycosylase interacts with A20R; essential for DNA replication 46 VACWR110(D5R) 66275907 785 1553 NTPase interacts with A20R; essential for DNA replication 57 VACWR127(A8R) 66275924 288 559 32kDa small subunit of transcription factor VITF-3 used for intermediate gene expression 62 VACWR138(A18R) 66275935 493 969 DNA helicase; effects elongation and termination of postreplicative viral transcription 65 VACWR143(A23R) 66275940 382 747 45kDa large subunit of intermediate gene transcription factor VITF-3 66 VACWR144(A24R) 66275941 1164 2311 DNA-dependent RNA polymerase subunit rpo132 68 A ORF K 70 123 69 VACWR154(A31R) 66275951 124 231 unknown 71 VACWR156(A33R) 66275953 185 353 EEV membrane phosphoglycoprotein associates with A36R; involved in CEV-cell adherence and actin tail formation 72 VACWR157(A34R) 66275954 168 319 EEV glycoprotein involved in CEV cell adherence and actin tail formation 73 VACWR158(A35R) 66275955 176 335 unknown 74 VACWR159(A36R) 66275956 221 425 IEV transmembrane phosphoprotein interacts with A33R and used in actin tail formation 78 VACWR167(A42R) 66275964 133 249 profilin-like 82 VACWR172(A46R) 66275969 240 463 Toll/IL1-receptor [TIR]-like; suppresses TIR-dependent signal transduction; host defense modulator 84 VACWR176(A50R) 66275973 552 1087 DNA 85 VACWR177(A51R) 66275974 334 651 unknown 86 VACWR178(A52R) 66275975 190 363 Toll/IL1-receptor [TIR]-like suppresses TIR-dependent signal transduction; host defense modulator 89 VACWR180(A55R) 66275977 564 1111 kelch-like 92 VACWR183(B1R) 66275980 300 583 ser/thr kinase essential for viral DNA replication 96 VACWR188(B6R) 66275985 173 329 ankyrin-like 98 VACWR190(B8R) 66275987 272 527 soluble interferon-gamma receptor-like; host defense modulator 100 VACWR192(B10R) 66275989 166 315 unknown 102 VACWR194(B12R) 66275991 283 549 ser/thr -like 103 VACWR195(B13R) 66275992 345 673 SPI-2/CrmA inhibits Fas-mediated apoptosis, IL-1 convertase, lipoxygenase pathway 105 VACWR197(B16R) 66275994 326 635 IL-1-beta-inhibitor [VACWR-B15R]; prevents febrile response in VAC-mouse intranasal model; host defense modulator 107 VACWR200(B19R) 66275997 351 685 IFN-alpha/beta-receptor-like secreted glycoprotein; host defense modulator [VACWR-B18R] 111 VACWR205(C12L) 66276002 353 689 serine protease inhibitor-like SPI-1; host defense modulator 113 VACWR010(C10L) 66275807 331 645 IL-1 receptor antagonist (Kluczyk, 2002, Peptides, 23:823) 114 VACWR008(C19L) 66275805 112 207 [ITR] gene fragment, ankyrin-like 118 VACWR001(C23L)/VACWR218(B29R)66275798/66276015 244 471 [ITR] chemokine-binding protein; host defense modulator 125 VACWR013 66275810 68 119 interleukin-18-binding protein; host defense modulator; absent from VACCP 129 VACWR019(C9L) 66275816 634 1251 ankyrin-like 131 VACWR021(C7L) 66275818 150 283 host-range; possible host defense modulator 132 VACWR022(C6L) 66275819 151 285 unknown 133 VACWR023(C5L) 66275820 204 391 unknown 134 VACWR024(C4L) 66275821 315 613 unknown 135 VACWR025(C3L) 66275822 263 509 secreted complement binding [C3b/C4b]; host defense modulator 137 VACWR027(C1L) 66275824 229 441 unknown 139 VACWR029(N2L) 66275826 175 333 alpha-amanitin target 140 VACWR030(M1L) 66275827 420 823 ankyrin-like 141 VACWR031(M2L) 66275828 220 423 unknown 143 VACWR033(K2L) 66275830 369 721 serine protease inhibitor-like; SPI-3, prevents syncytia; host range determinant; host defense modulator 144 VACWR034(K3L) 66275831 88 159 interferon resistance; host defense modulator 148 VACWR040(F1L) 66275837 226 435 localizes to the mitochondria and inhibits apoptosis by interfering with the release of cytochrome c (Stewart, 2005, J.Virol, 79:1084) 149 VACWR041(F2L) 66275838 147 277 dUTPase 151 VACWR043(F4L) 66275840 319 621 small subunit 152 VACWR044(F5L) 66275841 322 627 unknown 157 VACWR049(F10L) 66275846 439 861 ser/thr kinase 158 VACWR051(F12L) 66275848 635 1253 involved in plaque and EEV formation; on IEV, not CEV, IV or IMV 159 VACWR052(F13L) 66275849 372 727 palmytilated EEV membrane protein; phospholipase motif, required for IEV formation 163 E ORF A 70 123 165 VACWR058(E2L) 66275855 737 1457 unknown 166 VACWR059(E3L) 66275856 190 363 double-stranded RNA binding protein; inhibits antiviral activities of interferon; host-range determinant; host defense modulator 170 VACWR065(E9L) 66275862 1006 1995 DNA polymerase 171 VACWR067(E11L) 66275864 129 241 virion core protein 172 VACWR068(O1L) 66275865 666 1315 unknown 174 VACWR070(I1L) 66275867 312 607 DNA-binding core protein 176 VACWR072(I3L) 66275869 269 521 ssDNA-binding phosphoprotein 177 VACWR073(I4L) 66275870 771 1525 ribonucleotide reductase large subunit 178 VACWR074(I5L) 66275871 79 141 IMV protein VP13 179 VACWR075(I6L) 66275872 382 747 unknown 183 VACWR085(G7L) 66275882 371 725 virion structural protein 188 VACWR102(H4L) 66275899 794 1571 RAP94; tightly associated with DNA-dependent RNA polymerase, aids early-stage transcription preinitiation and termination 196 VACWR117(D12L) 66275914 287 557 small subunit of mRNA capping enzyme; transcription termination factor VTF 197 VACWR118(D13L) 66275915 551 1085 rifampicin target associates with inner surface immature virus membrane 199 VACWR120(A2L) 66275917 224 431 late gene transcription factor VLTF-3 201 VACWR122(A3L) 66275919 643 1269 p4b precursor of core protein 4b 204 VACWR125(A6L) 66275922 371 725 unknown 205 VACWR126(A7L) 66275923 710 1403 82kDa large subunit of early gene transcription factor VETF 207 VACWR129(A10L) 66275926 891 1765 precursor p4a of core protein 4a, complexes with A4L 208 VACWR131(A12L) 66275928 192 367 core protein 209 VACWR133(A14L) 66275930 90 163 phosphorylated IMV membrane protein required for morphogenesis 212 VACWR137(A17L) 66275934 203 389 IMV membrane protein undergoes phosphorylation and proteolytic processing; required for morphogenesis 213 VACWR139(A19L) 66275936 77 137 unknown 221 VACWR149(A26L) 66275946 213 409 gene fragment, cowpox A-type inclusion protein 229 VACWR162(A38L) 66275959 277 537 CD47-like putative membrane protein 234 VACWR173(A47L) 66275970 252 487 unknown 237 B ORF A 107 197 250 VACWR039(K7R) 66275836 149 281 unknown 252 VACWR064(E8R) 66275861 273 529 membrane protein may help wrap virosome; associates with IV/IMV and cores; F10L kinase substrate 254 VACWR082(G5R) 66275879 434 851 unknown 256 VACWR089(L2R) 66275886 92 167 unknown 258 VACWR184(B2R) 66275981 219 421 unknown 260 VACWR050(F11L) 66275847 348 679 unknown 263 VACWR090(L3L) 66275887 350 683 unknown, non-membrane protein packaged in mature virus particles. Role in infectivity.Essential gene.(Resch and Moss, J.Virol., 2005) 264 VACWR101(H3L) 66275898 324 631 IMV heparin binding surface protein involved in IMV maturation 269 VACWR170(A44L) 66275967 345 675 hydroxysteroid dehydrogenase; deletion attenuates intradermal lesion in VAC-mouse model; host defense modulator 270 VACWR174(A48R) 66275971 203 391

Total: 67150

Supplemental Table 1B. Binding affinities of known HLA class I epitopes

Binding capacity (IC50 NOTES Epitope Source organism MHC restriction1 nM)2 1. Restriction as reported at either the LANL-HIV database, SYFPEITHI YLLEMLWRL EBV A*0201 0.46 database, or the Immune Epitope Database (IEDB), or Bihl et al., The Journal LLWTLVVLL EBV A*0201 1.6 of Immunology, 2006, 176: 4094-4101. Only epitopes for which restriction LMPIIPLINV EBV A*0201 2.7 mapping had been done at the allele level were included. TLFIGSHVV EBV A*0201 15 SLVIVTTFV EBV A*0201 17 2. Exact redundancies were removed (i.e., reduced to a single entry), and the LLWAARPRL EBV A*0201 21 most "representative" example was selected. When duplicates differed as to CLGGLLTMV EBV A*0201 35 GLCTLVAML EBV A*0201 110 whether the peptide was a binder or not (500 nM used to define a binder), a VLQWASLAV EBV A*0201 247 geometric average was taken, and the replicate falling on the same side of the LLSAWILTA EBV A*0203 104 bind/not bind divide as the geometric average was taken as the representative SLREWLLRI EBV A*0203 4247 sample. LTAGFLIFL EBV A*0206 - AVFDRKSDAK EBV A*0301 59 Also, as much as could be identified, single (or double, when differences ATIGTAMYK EBV A*1101 2.1 were very conserved chemically) differences were treated as redundancies. IVTDFSVIK EBV A*1101 4.3 Peptides of 12 residues or longer were removed from both analysis sets, as in AVFDRKSDAK EBV A*1101 99 most cases it is very obvious that the correct minimal epitope was not AYSSWMYSY EBV A*3002 2.1 discerned. Also, where evidence indicates that a peptide is not the optimal AVFDRKSDAK EBV A*6801 99 epitope, the sequence was also removed. This occurs in some cases where a QPRAPIRPI EBV B*0702 3.5 nested or longer epitope has also been shown to be restricted by the same RPPIFIRRL EBV B*0702 3.9 VSFIEFVGW EBV B*5801 0.56 FLPSDFFPSV HBV A*0201 2.8 WLSLLVPFV HBV A*0201 4.8 STLPETTVVRR HBV A*3101 181 STLPETTVVRR HBV A*6801 26 SLLNATDIAV HIV A*0201 6.6 SLYNTVATL HIV A*0201 9.1 ALVEICTEM HIV A*0201 25 FLGKIWPSYK HIV A*0201 40 ILKEPVHGV HIV A*0201 101 AIIRILQQL HIV A*0201 522 VIYQYMDDL HIV A*0201 5995 PLTFGWCYKL HIV A*0201 24539 VLEWRFDSRL HIV A*0201 36749 SLYNTVATL HIV A*0202 2.9 ILKEPVHGV HIV A*0202 12 VIYQYMDDL HIV A*0202 48 QVPLRPMTYK HIV A*0301 0.50 TVYYGVPVWK HIV A*0301 0.54 RIRTWKSLVK HIV A*0301 1.1 KTKPPLPSVKK HIV A*0301 1.4 AIFQSSMTK HIV A*0301 1.4 HMYISKKAK HIV A*0301 2.6 RLRDLLLIVTR HIV A*0301 6.3 KIRLRPGGK HIV A*0301 25 RLRPGGKKK HIV A*0301 46 RLRPGGKKKY HIV A*0301 83 ALVEICTEMEK HIV A*0301 791 QIIEQLIKK HIV A*1101 1.4 AIFQSSMTK HIV A*1101 2.2 AVDLSHFLK HIV A*1101 2.6 QVPLRPMTYK HIV A*1101 2.7 SVITQACPK HIV A*1101 6.5 AVFIHNFKRK HIV A*1101 8.4 RYLKDQQLL HIV A*2301 0.85 KYKLKHIVW HIV A*2402 0.88 RYLKDQQLL HIV A*2402 5.4 RYPLTFGW HIV A*2402 17 LFCASDAKAY HIV A*2402 - EVIPMFSAL HIV A*2601 0.099 ETKLGKAGY HIV A*2601 32 RSLYNTVATLY HIV A*3002 0.29 KLNWASQIY HIV A*3002 0.54 HIGPGRAFY HIV A*3002 0.77 KQNPDIVIY HIV A*3002 4.8 KIQNFRVYY HIV A*3002 9.9 KYCWNLLQY HIV A*3002 10 IVNRNRQGY HIV A*3002 96 RMRGAHTNDV HIV A*3002 130 RLRDLLLIVTR HIV A*3101 3.8 ITKGLGISYGR HIV A*6801 13 AIFQSSMTK HIV A*6801 84 IVTRIVELL HIV A*6802 11 ITLWQRPLV HIV A*6802 23696 DTVLEEMNL HIV A*6802 - RPMTYKAAL HIV B*0702 0.88 FPVTPQVPL HIV B*0702 1.0 TPGPGVRYPL HIV B*0702 5.1 IPRRIRQGL HIV B*0702 8.3 TPQVPLRPM HIV B*0702 8.5 FPRIWLHGL HIV B*0702 13 HPRVSSEVHI HIV B*0702 14 SPRTLNAWV HIV B*0702 14 RPNNNTRKSI HIV B*0702 23 TPQDLNTML HIV B*0702 723 GPGHKARVL HIV B*0702 764 YPLTFGWCY HIV B*0702 - YLKDQQLL HIV B*0801 19 FPVTPQVPL HIV B*3501 0.50 HPDIVIYQY HIV B*3501 7.9 NPDIVIYQY HIV B*3501 9.0 NSSKVSQNY HIV B*3501 51 DPNPQEVVL HIV B*3501 1039 TAVPWNASW HIV B*3501 2242 NPVPVGNIY HIV B*3501 7945 VPVWKEATTTL HIV B*3501 10730 PPIPVGDIY HIV B*3501 11701 LPCRIKQII HIV B*5101 2244 YPLTFGWCY HIV B*5301 0.066 QASQEVKNW HIV B*5301 9.7 TPYDINQML HIV B*5301 14 EPVDPRLEPW HIV B*5301 54 TPQDLNTML HIV B*5301 203 ETINEEAAEW HIV B*5301 3076 KTAVQMAVF HIV B*5701 3.8 TSTLQEQIGW HIV B*5701 7.0 KAVRLIKFLY HIV B*5701 15 AVRHFPRIW HIV B*5701 18 KAFSPEVIPMF HIV B*5701 21 ISPRTLNAW HIV B*5701 44 IVLPEKDSW HIV B*5701 50 ISKKAKGWF HIV B*5701 122 QASQEVKNW HIV B*5701 144 IAMESIVIW HIV B*5801 0.73 TSTVEEQIQW HIV B*5801 1.2 TSTLQEQIGW HIV B*5801 3.7 KAVRLIKFLY HIV B*5801 5.5 IVLPEKDSW HIV B*5801 13 KAFSPEVIPMF HIV B*5801 35 QASQEVKNW HIV B*5801 62 ISPRTLNAW HIV B*5801 163 EADPTGHSY Human A*0101 44 LLGRNSFEV Human A*0201 38 GILGFVFTL Influenza A*0201 6.0 SRYWAIRTR Influenza B*2705 9.2