The eight vaccinated and control macaques were then challenged i.r. with 3.16 × 103
50% tissue culture infectious doses of SIVmac239/nef
) 6 weeks after receiving the MVA booster immunization. Mamu-A*01-positive animals were selected as controls, since Mamu-A*01-positive animals have demonstrated a lower viral set point following mucosal challenges with SIVmac251 (18
). Viral loads were quantitated with the SIVmac branched DNA (bDNA) RNA assay by the Bayer Reference Testing Laboratory (Emeryville, Calif.). Since we could find no evidence of viral replication in the Mamu-A*01-negative vaccinee (macaque 97007), it was omitted from further analysis. It is unlikely that this represents an example of sterilizing immunity against SIVmac239 challenge because occasional technical challenge failures have been observed after an i.r. challenge (although this is the first such failure in our hands using this stock of virus). Comparison of viral loads in the three remaining vaccinees to those of the controls revealed a slight, but not statistically significant, reduction in peak viremia (P
= 0.954; t
-test after log-transforming data to improve normality and homoscedasticity) and a slightly more pronounced diminution in viral loads at week 4 in the vaccinees. However, by week 10, viral loads in the vaccinees and controls were indistinguishable (P
= 0.954; t
-test) (Fig. ). This suggested that there might have been some short-term control of infection in the early weeks following challenge. Interestingly, however, there was a 1.5-log difference in viral loads of vaccinees verses controls at 1 week p.i., with the three vaccinees demonstrating higher viral loads at this early point in the infection.
FIG. 3. Viral loads of vaccinated and control macaques. Viral loads were quantitated from plasma by the Bayer Reference Testing Laboratory's SIVmac bDNA RNA Assay. Viral loads are indicated for each animal in the study, with the average viral loads of the vaccinees (more ...)
Following challenge with SIVmac239, there was a massive Tat28-35SL8-specific CTL recall response. In vaccinees, peak levels of 15.8, 28.8, and 7.3% were detected between 2 and 3 weeks p.i. (Fig. ). In comparison, in the control animals peak CTL levels were not achieved until 3 to 4 weeks p.i. Furthermore, these CTL responses in the controls (data not shown) were lower (between 2.0 and 8.5%) than those seen in the vaccinees (between 7.3 and 28.8%). Gag181-189CM9-specific acute-phase Mamu-A*01-restricted CTL responses were also measured. With the exception of control animal 85013, which exhibited levels of Gag181-189CM9-specific CTL of >6.0%, there was no difference in the peak levels of the Gag181-189CM9-specific CTL between vaccinees and controls. Interestingly, however, Gag181-189CM9-specific CTL appeared to peak earlier in the vaccinees, occurring at 3 weeks p.i. compared to 4 weeks p.i. in the controls.
In light of our previous discovery that the Tat28-35SL8 CTL response selects for escape variants during early infection, we reasoned that strong anamnestic Tat28-35SL8-specific CTL responses, coupled with high viral loads, would select for CTL escape variants in the vaccinees. Indeed, Tat28-35SL8 CTL escape mutants largely replaced wild-type virus in the plasma within 4 weeks p.i. in both the vaccinated and control animals (data not shown). This finding suggests that reduction of peak viremia to a level that does not support the emergence of escape variants may be critically important for vaccine regimens that include CTL epitopes that escape rapidly during natural infection.
Tat-specific proliferative responses were again measured at 2 and 6 weeks post-SIV infection (Fig. ). Robust proliferative responses were now detectable in the majority of vaccinees compared to only one of four controls at these time points tested. Interestingly, vaccinee 97007, the animal that did not become infected, continued to exhibit the highest levels of Tat-specific proliferative responses. This could represent either a boosting of the proliferative immune response from some low-level exposure to the virus during challenge or simply maintenance of the initial DNA/MVA-induced proliferative response.
These rather disappointing results should be interpreted with caution. We challenged vaccinated animals with a highly pathogenic molecular clone. With the exception of live-attenuated SIV (9
) or prior exposure to simian-human immunodeficiency virus clone 89.6 (15
), no vaccine regimen has been able to effectively control SIVmac239 replication. However, the SIVmac239 clone represents a good challenge virus to evaluate potential HIV vaccines since, like most primary HIV type 1 isolates, this virus is highly resistant to antibody-mediated neutralization (6
) and causes a gradual depletion of CD4 T lymphocytes. Furthermore, challenge of rhesus macaques with SIVmac239 yields reproducible viral set points of approximately 106
viral copies/ml, thus facilitating clear identification of vaccine efficacy. Additionally, despite our induction of high levels of Tat-specific CTL, we may not have induced these CTL at important mucosal sites. Furthermore, induction of a CTL response against a single CTL epitope may be similar to the use of single antiretroviral drug therapy, and induction of immune responses against multiple CTL epitopes may prove more effective, possibly analogous to the situation with combination drug therapy. Finally, while this vaccine induced strong CTL responses, the regimen was not designed to similarly induce strong CD4 T-cell helper responses, which might play an important role in the containment of HIV and SIV infections.