 evaluate changes in viral load as a result of in vivo CD8 ϩ T cell depletion, a novel assay was first developed to allow simultaneous quantification of both the vaccine strain (SIVmac239⌬nef) and the challenge virus (SIVmac251) in the same sample. This assay is based on real-time PCR using molecular beacons and primers hybridizing within and flanking the 182-bp deleted region of nef, and allows for discrimination of nef-deleted and wild-type SIV nef genes (Fig. 1, A and B). Initial evaluation of standard SIV RNA preparations defined a dynamic range of Ͼ5 logs for both nef-deleted and wild-type RNA when analyzed separately.
The discriminatory ability of the assays was tested in reciprocal mixing experiments by adding to a serial dilution (10 8 to 10 copies) of either SIVmac239⌬nef or SIVmac251 RNA or DNA standard, 10 8 copies of noncomplimentary RNA or DNA. Amplifications were performed and results were compared with threshold cycles from PCR reactions performed without the addition of noncomplimentary viral RNA or DNA. Results from these experiments indicate that the assay for amplification of SIVmac239⌬nef has a discriminatory ability of 1 copy of SIVmac239⌬nef/10 6 copies of SIVmac251, whereas the amplification of SIVmac251 has a discriminatory ability of 1/10 7 (Fig. 2, A and B).
Longitudinal plasma samples obtained from a rhesus macaque immunized with SIVmac239⌬nef but not protected from SIVmac251 challenge were then tested to determine the sensitivity and discriminatory ability of the assay under more biologically relevant conditions. The results demonstrate detection of both SIVmac239⌬nef and SIVmac251 in the same plasma sample with a sensitivity of Ն50 SIV RNA copies/ml, and document a decline in SIVmac239⌬nef replication that occurred temporally with an increase in replication of SIVmac251 (Fig. 2 C).
In further experiments, plasma samples from an unrelated rhesus macaque infected with wild-type SIV were also quantified in parallel using either real-time PCR or the Chiron bDNA assay, and the results were found to be highly similar (within threefold; data not shown), providing a basis for comparison with other quantitative SIV studies, including our previous report (3).
Using the real-time PCR assay to monitor changes in viral load, the anti-CD8 antibody OKT8F was administered to 4/6 rhesus macaques (1496, 1502, 1518, and 1520) at a concentration of 2 mg/kg/d for three consecutive days. The two remaining macaques (1506 and 1522) received an isotype-matched control antibody, P1.17. Blood samples taken at frequent intervals before and after administration of the antibodies were assayed to monitor changes in CD8 ϩ T cells and viral load (Fig. 3). Within 1 h after administration of the first dose of antibody, CD8 ϩ T cells decreased in the peripheral blood by an average of 99% (from a mean of 1,151 Ϯ 428 to 0 cells/ml) in the four macaques receiving OKT8F, whereas the CD8 ϩ T cell counts remained stable in the two macaques receiving the control antibody (1506 and 1522). In error, OKT8F was given at the second injection to one of the control macaques (1522), resulting in a similar, rapid depletion of CD8 ϩ T cells (Fig. 3 F). To evaluate the effect of a single dose of OKT8F, the control antibody was then administered to macaque 1522 for the third injection. For the four macaques receiving three doses of OKT8F, the number of CD8 ϩ T cells remained low for 8-9 d, and began to re- A linearized upstream primer (nef1) and hairpin-shaped primer (hs ⌬nef) were used to amplify a 106-bp amplicon. hs ⌬nef hybridizes across the junction of the deletion in nef, allowing amplification of SIVmac239⌬nef in the presence of SIVmac251. A molecular beacon (mb ⌬nef) was used for detection of amplicons. (B) Amplification of SIVmac251. A linearized primer (nef1) and hairpin-shaped oligonucleotide (hs wt nef) were used to amplify a fragment of 142 bp. An additional PCR was performed using linearized oligonucleotides (nef3 and wt nef2), which amplify a fragment of 157 bp. Amplicons were detected using the molecular beacon (mb wt nef). cover in all animals by day 10. A slightly more rapid recovery of CD8 ϩ T cells, occurring on day 8, was observed for the macaque receiving one dose of OKT8F, suggesting a less sustained depletion.
Concomitant with the decrease in peripheral blood CD8 ϩ T cells was a dramatic increase in the levels of SIV-mac239⌬nef in plasma, which occurred in 5/5 macaques receiving OKT8F antibody. Viral loads at baseline were low in all immunized macaques (0.22-6.1 ϫ 10 3 SIV RNA copies/ml), but increased by a minimum of 1-2 logs after CD8 ϩ T cell depletion. Viremia reached a mean peak value of 1.7 ϫ 10 5 SIV RNA copies/ml (0.067-6.4 ϫ 10 5 copies/ml) between days 1 and 8 after antibody administration. Moreover, the decrease in SIVmac239⌬nef viremia was temporally associated with recovery of the CD8 ϩ T cell population in all macaques. Although fluctuations in both CD8 ϩ T cells and viral load occurred in the one macaque receiving three doses of the control antibody, there was no clear temporal association between these changes and administration of the antibody (Fig. 3 C), similar to what has been observed using the same control antibody in rhesus macaques chronically infected with SIV (29).
SIVmac239⌬nef proviral DNA at baseline was low or undetectable (Ͻ50 copies/10 6 genomic equivalents) in rhesus macaques receiving OKT8F and increased by 0.6-2 logs after administration of OKT8F. Interestingly, the increase in SIVmac239⌬nef proviral DNA occurred 1-3 d after the increase of viral RNA in plasma. Peak values for proviral DNA ranged from 3 ϫ 10 2 to 3.1 ϫ 10 4 copies/ 10 6 genomic equivalents. A decrease of proviral DNA was temporally associated with the increase of CD8 ϩ T cells. Changes in SIVmac239⌬nef proviral load in rhesus macaque 1506 were not associated with administration of the control antibody (Fig 3 C).
Simultaneous quantification of SIVmac239⌬nef and SIVmac251 was carried out using plasma and PBMC sam- Amplifications of SIVmac239⌬nef were performed using a serial dilution of SIVmac239⌬nef DNA standard with (᭺) and without (ᮀ) the addition of 10 8 copies of noncomplimentary SIVmac251 DNA standard. The linear standard curve for amplification of SIVmac239⌬nef DNA alone is shown in black. The gray section indicates the area where the ratio of copies of SIVmac239⌬nef to copies of SIVmac251 is Ն1/10 6. (C) Measurement of SIV RNA in plasma after immunization with SIVmac239⌬nef and challenge with SIVmac251. Rhesus macaque 1510 was immunized with SIVmac239⌬nef on day 0 (black arrow) and then challenged with SIVmac251 at 25 wk (white arrow). SIVmac239⌬nef RNA (᭹) and SIVmac251 RNA () in plasma were quantified using real-time PCR for differential amplification. The limit of sensitivity of the viral load assay is indicated by the dotted line. Changes in viral load after in vivo depletion of CD8 ϩ T cells. SIV RNA in plasma and SIV proviral DNA in PBMCs were determined for each sample in duplicate by real-time PCR and differential amplification. SIV proviral DNA data were normalized per 10 6 genomic equivalents measured of the single-copy gene CCR5. Data are shown for CD8 ϩ T cells (᭹), SIVmac239⌬nef RNA (), and SIVmac239⌬nef proviral DNA (ᮀ). SIVmac251 RNA or DNA were not detectable. Administrations of OKT8F (black arrows) or the control antibody P1.17 (white arrows) were performed on days 0, 1, and 2. The limit of sensitivity of the viral load assay is indicated by the dotted line. ples from the three macaques that were immunized and challenged (1496, 1502, and 1506; Table I). In an earlier study using conventional DNA PCR, we were unable to detect wild-type nef sequences in PBMCs from these macaques over a period of 2 yr after challenge (Connor, R., unpublished data). Before depletion of CD8 