aSupplemental Table 1: Gamma-Interferon ELISPOT Responses Post-Immunization
Antigen-specific IFN- ELISPOT 6a(spot forming cells/10 PBMC)
Prior to boost immunization Post boost immunization
Vaccine Animal SIV gag SIV gag c c pool pool Group Regimen ID Mock p11CM Mock p11CM
Study A b 1 Naïve 115Q n.t.n.t. n.t. n.t. n.t. n.t.
control V388 8 6 4 19 16 n.t.
2 DNA 97X011 24 34 59 23 59 n.t.
T271 25 159 65 13 273 n.t. a T28211 23 59 111 289 n.t.
3 DNA + S202 10 858 88 10 1261 n.t.
CRL 047R 16 369 139 15 319 n.t.
079R 18 140 219 1 224 n.t.
4 DNA + 058R 18 195 81 61 70 n.t.
MPL/Alum 088R 9 61 53 28 251 n.t.
108R 8 265 70 24 108 n.t.
5 MVA 122G 123 85 105 55 146 n.t.
126G 130 123 170 118 193 n.t.
15G 148 198 180 36 140 n.t.
6 Ad5 54G 8 470 158 6 508 n.t.
56G 0 928 305 16 435 n.t.
82F 8 835 108 8 314 n.t.
7 Naïve 89Q 5 8 0 n.t. n.t. n.t.
8 DNA/CRL 98D273 0 58 25 0 1238 188
+ 98D318 5 963 186 0 4605 373
MVA boost 98D359 0 1113 159 0 1955 683
9 DNA/CRL 98C040 0 338 66 0 17680 623
+ 98C081 0 329 71 0 14850 783
Ad5 boost 98C084 3 276 121 0 10090 1158 a ELISPOT assays were performed as described in text. The values reported here were obtained using
peripheral blood mononuclear cell (PBMC) preparations from bleeds prior to and four weeks post the boost
immunization. b Each of the PBMC pools was stimulated with either a mock peptide solution (mock) or with the p11CM
peptide alone (p11CM) or with a pool composed of overlapping peptides that spanned the entire SIV gag
region with the exception of the p11CM determinant (SIV gag pool).
ELISPOT assay: Sterile 96-well microtiter plates (PVDF, MAIP S45, Millipore)
owere coated overnight at 4C with 100 ?l/well of mouse anti-human IFN-? monoclonal
antibody (R&D Systems, Minneapolis, MN). The antibody was diluted to 10 ?g/ml in
osterile PBS. Plates were washed four times with sterile PBS and blocked 2 hours at 37C with 200 ?l/well of R10 complete medium (RPMI-1640 plus 10% heat-inactivated fetal
bovine serum). The blocking buffer was decanted and 100 ?l/well of rhesus PBMC
55diluted in R10 were added for for duplicate samples at 2x10 and 1x10 cells/well. Synthetic peptides were used for SIV gag antigen (see legend to Supplemental Table 1
for details) and were added to the wells at a final concentration of 2.5 ?g/ml per peptide. Peptide-free DMSO diluent matching the DMSO concentration in the peptide solutions
was used as a negative control (mock). Plates were incubated 24 hours in a humidified
oCO incubator at 37C and then washed 7 times with PBS containing 0.05% Tween 20 2
(Sigma). Biotinylated goat anti-human IFN-? monoclonal antibody (R&D Systems
catalog #BAF285), diluted to 0.1 ?g/ml in assay diluent consisting of PBS and 5% heat
inactivated fetal bovine serum (FBS) and 0.005% Tween 20, was added to the plates at
o100 ?l/well and incubated overnight at 4C. After washing 7 times with PBS/Tween, 100
?l/well of alkaline phosphatase-conjugated streptavidin (BD PharMingen, San Diego, CA) at 1:2500 in assay diluent was added to each well. Plates were incubated 2 hours at room
temperature and washed 7 times with PBS/Tween. To develop the spots, 100 ?l/well of alkaline phosphatase substrate NBT/BCIP (Pierce) was added to each well and incubated
at room temperature until spots became visible, usually 5-10 minutes. Spots were
counted with a stereomicroscope using a magnification of 20-25X. The number of spots
6per well was normalized per 1x10 cells and averaged for each sample and antigen based
upon the replicate samples for both of the two cell input levels.
The IFN-? ELISOT assay was formally validated to allow quantitative
discrimination of positive vs. negative responses as well as to determine the variability of the assay. Briefly, the validation protocol consisted of two basic parts: (1) testing PBMC samples obtained from animals that had never been exposed to SIV or HIV antigens using the gag peptide pools to establish the background response levels of negative subjects; (2) testing PBMC samples from positive subjects with the same peptide pools. The results from these studies led us to adopt a two part definition of a positive ELISPOT response: having at least 55 spots per million PBMCs treated with gag antigen, and having at least four-fold more spots for PBMCs treated with antigen compared to PBMCs treated with mock solution. When these two criteria are met there is greater than 99% probability that the test sample was no longer negative. Finally, the overall variability of the assay observed from positive samples was less than two-fold.
Supplemental Figure Legend
+ T cell responses by tetramer analysis Supplemental Figure 1: Quantitation of CD8
following SHIV challenge. Study A and Study B immunization groups are shown at the
++top and bottom, respectively. The percentage of CD3 CD8 cells that bind tetramer are
shown for each vaccine group. The SHIV challenge was administered intravenously at day 0.