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Supplemental Digital Content Koopman et al.
Supplemental Digital Content Fig. 1 SIVconsv immunogen and experimental design. A Schematic representation of the SIVconsv immunogen, showing the arrangement of the 14 conserved regions of SIV within the DNA.SIVconsv construct, peptide pool derivation and protein origin by grey scale coding. B Study schedule. Three groups of six animals were immunized with either HPV18 SLPs (group 1) or SLP.SIVconsv (S) (group 2) at week 4, 14, 36, while group 3 received a DNA.SIVconsv (D) prime at week 0 and 4 followed by a SLP.SIVconsv (S) boost at week 14 and 36. All animals were given a single high dose intrarectal SIVmac 251 challenge at week 44. Two naïve control animals (group 4) received only SIVmac251 challenge. Dose (μg used of each peptide) is indicated in parenthesis.
region 1
SIVconsv IEEKKFGAEVVPGFQALSEGCTPYDINQMLNCVGDHQAAMQIIRDII
HIVconsv M---A-SP--I---T-----A--Q-L-T---T--G------MLK-T-
region 2
SIVconsv NEEAADWDL PVGNIYRRWIQLGLQKCVRMYNPTNILDVKQGPKEP
HIVconsv -----E--R --K---I---N-I----S-VS---IR------
region 3
SIVconsv FQSYVDRF ARQCRAPRRQGCWKCGKMDHVMAKCPDRQAGFLGLG
HIVconsv -RD------N-----KK------EG-Q-KD-TE---N---KI
region 4
SIVconsv PWG ELGPHYTPKIVGGIGGFINTKEYKNVEIEVLGKRIKGTIMT
HIVconsv WPS RWK--MI------KVRQYDQIL--IC-HKAI--VLV
SIVconsv GDTPINIFGRNLLTALGMSLNFPIAKVEPVKVALKPGKDGPKLKQWPL
HIVconsv -P--V--I------QI-CT-----SPI-T-P-K----M----V-----
SIVconsv SKEKIVALREICEKMEKDGQLEEAPPTNPYNTPTFAIKKKDKNKW
HIVconsv TE---K--V---TE---E-KISKIG-E------V------ST--
region 5
SIVconsv RMLIDFRELNRVTQDFTEVQLGIPHPAGLAKRKRITVLDIGDAYFSIP
HIVconsv -K-V------KR----W------K-K-SV----V------V-
SIVconsv LDEEFRQYTAFTLPSVNNAEPGKRYIYKVLPQGWKGSPAIFQYTMRHV
HIVconsv ---G--K-----I--I--ET--I--Q-N------SS-TKI
region 6
SIVconsv LEPFRKANPDVTLVQYMDDILIASDRTDLEHDR MEEEKRWIAVP
HIVconsv -----AQ--EIVIY-----LYVG--LEIGQ-R --NRWQVMI-
region 7
SIVconsv TWRIPERLERWHSLIKYLK LTEEVQWTEMAEAEYEENKIILSQEQ
HIVconsv WQVDRM-IRT-K--V-HH ----AELELAENR-ILKDPVHGVYYD
region 8
SIVconsv EGCYYQEGKP YWQVTWIPEWDFISTPPLVRLVFNLVK
HIVconsv PSKDLIAEIQ ---A------E-VN-----K-WYQ-E-
region 9 region 10
SIVconsv NVTESFDAWENTVTEQAIEDVWQLFETSIKHCVKLSP WVPAHKGI
HIVconsv ----N-NM-K-DMVD-MH--IIS-WDQ-L-P----T------
region 11
SIVconsv GGNQEIDHLVSQGIRQVLFLEKIEPAQ ARQIVDTCDKCHQKGEA
HIVconsv ---EQV-K------K----DG-DK-- -KE--AS----QL----
SIVconsv IHGQVNSDLGTWQMDCTHLEGKIIIVAVHVASGFIEAEVIPQETGRQT
HIVconsv M----DCSP-I--L------V-L------Y------A---QE-
region 12
SIVconsv ALFLLKLA MNHHLKNQIDRIREQANSVETIVLMAVHCMNFKRRG
HIVconsv -Y------KE--KI-GQV-D--EHLK-A-Q---FIH----K-
region 13
SIVconsv GIGDMTPAERL WKGPGELLWKGEGAVILKVGTDIKVVPRRKAKI
HIVconsv ---GYSAG--I ----AK------VIQDNS------
region 14
SIVconsv IKDYGGGKEVDSSS FLATAGSAMGAASLTLTAQSRTLLAGIVQQ
HIVconsv -R---KQMAGADCV --GA---T-----M---V-ARQ--S-----
SIVconsv QQQLLDVVKRQQELLRLTVWGTKN
HIVconsv -NN--RAIEA--H--Q-----I-Q
Supplemental Digital Content Fig. 2. Amino acid sequence of the 14 SIVconsv regions and alignment with the HIVcons sequences. The 14 regions are represented as separate stretches with the region number indicated above the sequence. Underlined are amino acids that were inserted to allow better production of the peptide.
Supplemental Digital Content Fig. 3 Representative example of cytokine expression in CD4+ and CD8+ T-cell subsets. Shown is for animal R06062, the position of pp6 specific IFN-γ, IL-2 and TNFα producing cells (white dots) within the CD28/CD95 contour plot (in black, formed by plotting the total CD4+ or CD8+ T-cell population). Indicated on top of each graph is the total percentage of positive cells for each cytokine. Numbers inside the graphs indicate the relative distribution of antigen-specific cytokine-producing cells over each of the four quadrants.
Supplemental Digital Content Table 1. TRIM5α and MHC class I genotypes.
group / animals / TRIM5 / MHC class I1naïve / R03099 / TFP/TFP / A1*008, B*036, B*037/A1*002, B*001, B*007
controls / R05054 / Q/Q / A1*008, B*036, B*037/A1*002, B*001, B*007
HPV / R02024 / TFP/Q / A1*004, B*015, B*044/A1*008, B*038, B*047
controls / R02080 / TFP/TFP / A1*008, B*015, B*044/A1*002, B*001, B*007
R04060 / TFP/CYP / A1*007, B*019, B*024/A1*003, B*001, B*007
R04111 / Q/Q / A1*004, B*012, B*038/A1*007, B*019, B*024
R05016 / Q/Q / A1*002, B*001, B*007/A1*002, B*001, B*007
R06019 / Q/Q / A1*002, B*001, B*007/A1*026, B*012, B*022
SSS / 98032 / TFP/Q / A1*004, B*001, B*007/A1*006, B*043, B*027
R01087 / TFP/Q / A1*004, B*012, B*038/A*003, A2*24, B*001, B*007
R02013 / TFP/Q / A1*004, B*012, B*022/A1*006, B*001, B*007
R05086 / TFP/Q / A1*003, B*001, B*007/A1*004, B*015, B*044
R06016 / Q/Q / A1*002, B*001, B*007/A1*026, B*012, B*022
R06080 / Q/Q / A1*004, B*001, B*007/A1*008, B*028, B*021
SSDD / R00007 / TFP/Q / A1*004, B*012, B*038/A*003, A2*24, B*001, B*007
R02044 / TFP/CYP / A1*004, B*?/A1*006, B*043, B*027
R04045 / TFP/Q / A1*004, B*015, B*044/A1*008, B*038, B*047
R04066 / Q/Q / A1*004, B*001, B*007/A1*004, B*012, B*022
R04096 / TFP/Q / A*003, A2*24, B*036, B*037/A1*007, B*001, B*007
R06062 / TFP/Q / A1*023, B*012, B*022/A1*004, B*024, B*019
1shown is paternal and maternal MHC class I haplotype, separated by a slash.
Supplemental Digital Content Table 2 Peptide responses detected in the IFNγ ELISpot assay. ICS was used to determine whether responses were CD4+ of CD8+ T-cell mediated.
Group / Animal / pp / Region / Peptide sequence / CD4/8HPV SLP / R04060 / HPV / CHKCIDFYSRIRELR / CD4
control / HPV / IRCLRCQKPLNPAE / CD4
HPV / LRCQKPLNPAEKLRH / CD4
5 / POL11 / IIVAVHVASGFIEAEKKKK / CD4
SIVconsv / 98032 / 5 / POL11 / HVASGFIEAEVIPQE / CD4
SSS / 5 / POL11 / FIEAEVIPQETGRQT / CD4
5 / POL11 / VEIPQETGRQTALFL / CD4
6 / ENV14 / RTLLAGIVQQQQQLL / CD4
R01087 / 5 / POL11 / LEGKIIIVAVHVASG / CD8
R02013 / 1 / GAG2 / KCVRMYNPTNILDVK / CD4
3 / POL5 / FTEVQLGIPHPAGLA / n.d
3 / POL5 / LGIPHPAGLAKRKRI / n.d.
3 / POL5 / KRKRITVLDIGDAYF / n.d
3 / POL5 / TVLDIGDAYFSIPLD / n.d.
3 / POL5 / EEFRQYTAFTLPSVN / CD8
3 / POL5 / AIFQYTMRHVLEPFR / CD8
3 / POL5 / QYTMRHVLEPFRKAN / CD8
3 / POL5 / VLEPFRKANPDVTLV / CD8
5 / POL11 / HVASGFIEAEVIPQE / CD4
5 / POL11 / FIEAEVIPQETGRQT / CD4
6 / POL13 / ILKVGTDIKVVPRRK / CD4
6 / POL13 / RKAKIIKDYGGGKEV / CD4
R05086 / 3 / POL5 / FTEVQLGIPHPAGLA / n.d.
3 / POL5 / LGIPHPAGLAKRKRI / n.d.
3 / POL5 / AIFQYTMRHVLEPFR / n.d.
4 / VIF6 / RWIAVPTWRIPERLE / CD4
4 / ENV9 / DVWQLFETSIKHCVK / CD4
5 / POL11 / HVASGFIEAEVIPQE / CD4
6 / POL12 / TIVLMAVHCMNFKRR / CD4
R06016 / 3 / POL5 / KRYIYKVLPQGWKGS / CD4
R06080 / 3 / POL5 / FTEVQLGIPHPAGLA / CD8
3 / POL5 / LGIPHPAGLAKRKRI / CD8
3 / POL5 / PAGLAKRKRITVLDI / CD4
5 / POL11 / EAIHGQVNSDLGTWQ / CD4
5 / POL11 / HVASGFIEAEVIPQE / CD4
6 / ENV14 / RTLLAGIVQQQQQLL / CD4
SIVconsv / R00007 / 1 / GAG2 / YRRWIQLGLQKCVRM / CD4
DDSS / 5 / POL11 / FIEAEVIPQETGRQT / CD4
5 / POL11 / VIPQETGRQTALFL / CD4
6 / POL13 / TDIKVVPRRKAKIIK / CD4
6 / POL13 / RKAKIIKDYGGGKEV / CD4
6 / ENV14 / QQQLLDVVKRQQELL / CD4
6 / ENV14 / DVVKRQQELLRLTVW / CD4
6 / ENV14 / RQQELLRLTVWGTKN / CD4
R02044 / 3 / POL5 / AIFQYTMRHVLEPFR / CD4
5 / POL11 / HVASGFIEAEVIPQE / CD4
5 / POL11 / FIEAEVIPQETGRQT / CD4
6 / ENV14 / RQQELLRLTVWGTKN / CD4
R04045 / 5 / POL11 / HVASGFIEAEVIPQE / CD4
5 / POL11 / FIEAEVIPQETGRQT / CD4
R04066 / 5 / POL11 / HVASGFIEAEVIPQE / CD4
5 / POL11 / FIEAEVIPQETGRQT / CD4
6 / POL13 / WKGPGELLWKGEGAV / CD4
6 / POL13 / ELLWKGEGAVILKVG / CD4
R04096 / 5 / POL11 / IIVAVHVASGFIEAEKKKK / CD4
5 / POL11 / HVASGFIEAEVIPQE / CD4
R06062 / 1 / GAG2 / YRRWIQLGLQKCVRM / CD4
1 / GAG2 / KCVRMYNPTNILDVK / CD4
1 / GAG2 / YNPTNILDVKQGPKE / CD4
4 / VIF6 / MEEEKRWIAVPTWRI / CD4
4 / VIF6 / RWIAVPTWRIPERLE / CD4
5 / POL10 / NQEIDHLVSQGIRQV / CD4
5 / POL10 / HLVSQGIRQVLFLEK / CD4
5 / POL11 / HVASGFIEAEVIPQE / CD4
5 / POL11 / FIEAEVIPQETGRQT / CD4
6 / ENV14 / RQQELLRLTVWGTKN / CD4
Previously published SIV CTL epitope is underlined and SIV CD4 epitope is bold and underlined. n.d. was not determined because of too low responses.
Supplemental Digital Content Table 3. Overview immunogenicity data in relation to peak and steady state virus load. Shown are for the SSS immunized animals of group 2 and the DDSS immunized animals of group 3 semi-quantitatively the IFNγ ELISpot responses measure at 2 weeks before challenge, ICS CD4 and CD8 total cytokine response measured after the second immunization, serum Ab titer against gp130 at 2 weeks before challenge and the mucosal IgG Ab response against SIV lysate measured 3 weeks before challenge. Virus load is expressed as RNA equivalents/ml plasma measured at 2 and 12 weeks after challenge.
group / animals / IFNγ ELISpot1 / ICS CD42 / ICS CD82 / Serum Ab titer SIVenv3 / Mucosal IgG Ab OD4054 / peak VL (wk 2) / steady state VL (wk 12)SSS / 98032 / +++ / ++ / - / + / - / 1,9E+07 / 7,8E+06
R01087 / ++ / ++ / + / + / ++ / 6,3E+07 / 1,6E+07
R02013 / +++ / +++ / ++ / + / ++ / 8,0E+06 / 2,0E+06
R05086 / ++ / ++ / ++ / ++ / - / 1,8E+07 / 3,0E+06
R06016 / ++ / + / - / + / + / 8,3E+06 / 8,3E+05
R06080 / ++ / ++ / ++ / - / - / 1,0E+07 / 5,2E+05
SSDD / R00007 / + / + / - / + / +++ / - / -
R02044 / +++ / ++ / - / + / ++ / 9,7E+05 / 1,7E+04
R04045 / ++ / + / - / - / + / 3,3E+06 / 3,0E+06
R04066 / + / ++ / - / + / + / 3,5E+07 / 4,8E+07
R04096 / ++ / ++ / - / + / +++ / - / -
R06062 / +++ / +++ / + / ++ / - / 1,2E+07 / 5,9E+06
1ELISpot responses: + 100-1000, ++ 1000-3000, +++ > 3000 SFU/106 PBMC
2ICS responses: - no response, + <1%, ++ 1-5%, +++ >5% specific cytokine expressing cells as percentage of CD4 or CD8 T-cells
3Serum Ab titer: - no response, + 1:400 or less, ++ more than 1:400 dilution positive
4Mucosal IgG Ab titer: - no response, + <0.15, ++ 0.15-0.30, +++ > 0.30 OD405/μg total IgG.