Protective Efficacy of a Single Immunization of a Chimeric Adenovirus Vector-Based Vaccine against Simian Immunodeficiency Virus Challenge in Rhesus Monkeys

We monitored vaccine-elicited, SIV-specific cellular immune responses prior to challenge by gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assays following stimulation with SIV Gag, Pol, Nef, and Env peptide pools (8). As shown in Fig. 1A, monkeys that received a single immunization of the rAd vaccines alone at week 24 developed IFN-γ ELISPOT responses to the encoded SIV antigens by week 26. As expected, animals that were primed with the DNA vaccines developed higher responses than those elicited by the rAd alone vaccines (16, 18). The adjuvanted DNA vaccines elicited a trend toward higher peak responses compared with the unadjuvanted DNA vaccines, but these differences were not sustained at week 52.

As shown in Fig. 1B, DNA priming augmented both CD8⁺ and CD4⁺ T-lymphocyte responses, as determined by cell-depleted IFN-γ ELISPOT assays (P = 0.01 and P = 0.001, respectively [Wilcoxon rank-sum tests]). Multiparameter intracellular cytokine staining (ICS) assays (8, 13, 14) confirmed these findings but did not reveal other consistent phenotypic differences in terms of IFN-γ, tumor necrosis factor alpha (TNF-α), and interleukin 2 (IL-2) secretion in CD8⁺ and CD4⁺ central memory (CM [CD28⁺ CD95⁺]) and effector memory (EM [CD28⁻ CD95⁺]) T-lymphocyte subpopulations (Fig. 1C). The ICS assays utilized SIV Gag, Pol, Nef, and Env peptide pools and the following monoclonal antibodies: anti-CD3-Alexa700 (SP34), anti-CD4-AmCyan (L200), anti-CD8-allophycocyanin-Cy7 (anti-CD8-APC-Cy7) (SK1), anti-CD28-PerCP-Cy5.5 (L293), anti-CD95-phycoerythrin (anti-CD95-PE) (DX2), anti-IFN-γ-PE-Cy7 (B27), anti-IL-2-APC (MQ1-17H12), and anti-TNF-α-fluorescein isothiocyanate (anti-TNF-α-FITC) (Mab11).

Six months after the rAd boost immunization, monkeys were challenged i.v. with the essentially homologous virus SIVmac251 (7, 10). As shown in Fig. 2A, all vaccinated animals developed rapid and potent anamnestic SIV-specific cellular immune responses by week 2 following challenge, as determined by IFN-γ ELISPOT assays. SIV-specific humoral immune responses were evaluated by luciferase-based pseudovirus neutralization assays (12) and antibody-dependent cell-mediated virus inhibition (ADCVI) assays (5). All animals developed virus-specific NAbs against laboratory-adapted SIVmac251 but not against primary isolate SIVmac251 following challenge (Fig. 2B). Animals that received vaccines containing SIV Env (groups 1 to 3) also developed more rapid kinetics of ADCVI than animals that received sham controls or vaccines that did not include Env (Fig. 2C).

We next monitored the CD4⁺ T-lymphocyte dynamics in these animals following challenge (10) utilizing the following monoclonal antibodies: anti-CD3-Alexa700 (SP34), anti-CD4- AmCyan (L200), anti-CD8-APC-Cy7 (SK1), anti-CD28-PerCP-Cy5.5 (L293), anti-CD95-APC (DX2), anti-CCR5-PE (3A9), anti-HLA-DR-PE-Cy7 (L243), and anti-Ki67-FITC (B56). As shown in Fig. 3, control animals exhibited slightly decreased levels of CD4⁺ CM T lymphocytes, substantially decreased levels of CCR5⁺ CD4⁺ CM T lymphocytes, and markedly increased levels of Ki67⁺ proliferation of CCR5⁺ CD4⁺ CM T lymphocytes following SIV challenge, consistent with our prior results (10). In contrast, animals vaccinated with rAd alone had fewer dramatic perturbations of these CD4⁺ T-lymphocyte subsets.

To assess the protective efficacy of these vaccine regimens, we monitored SIV RNA levels and survival in these animals following challenge, as depicted in Fig. 4. SIV RNA levels for each individual animal (Fig. 4A) as well as peak (day 14) (Fig. 4B) and set point (median days 112 to 392) (Fig. 4C) SIV RNA levels are shown. The rAd alone vaccine expressing only Gag, Pol, and Nef afforded only minimal control of SIV replication, but the rAd alone vaccine expressing Gag, Pol, Nef, and Env resulted in a 1.34 log reduction of peak SIV RNA levels (Fig. 4B) (P = 0.001 [Wilcoxon rank-sum test]) and a 1.08 log reduction of set point SIV RNA levels compared with sham controls (Fig. 4C) (P = 0.05). These data suggest that the addition of Env improved control of SIV replication following challenge in this study. The rAd alone vaccines also afforded trends toward improved survival compared with sham controls for 500 days following the challenge (Fig. 4D) (P = 0.04 and P = 0.07 [unadjusted two-sided log-rank tests comparing rAd-Gag/Pol/Nef/Env and rAd-Gag/Pol/Nef regimens, respectively, versus sham controls]). Moreover, in a prespecified exploratory analysis, the combined rAd alone vaccinated animals (n = 12) exhibited significantly improved survival compared with the sham controls (P = 0.003 [two-sided log-rank test]).

The monkeys that were primed with DNA vaccines or adjuvanted DNA vaccines prior to the rAd immunization, however, did not exhibit improved protection compared with the animals that received rAd alone vaccines. The DNA/rAd regimens afforded 0.94 to 1.11 log reductions of peak SIV RNA levels (Fig. 4B) (P = 0.004 [Wilcoxon rank-sum test]) but no significant reductions of set point SIV RNA levels (Fig. 4C) (P = nonsignificant) and only marginal improvements of survival (Fig. 4D) (P = nonsignificant) compared with the sham controls. These data show that DNA vaccine priming did not improve the protective efficacy afforded by rAd5HVR48 vectors in this study. These results are consistent with previous SIV challenge studies that have similarly shown no significant and durable benefit of DNA vaccine priming prior to rAd5 boosting in terms of set point SIV RNA levels or survival following challenge (2, 7). Casimiro et al. (2) reported that neither DNA/rAd5 regimens nor rAd5 alone regimens expressing SIV Gag resulted in significant reductions of set point SIV RNA levels following SIV challenge of Mamu-A*01-positive rhesus monkeys, although a transient 0.8 log reduction of peak SIV RNA levels was noted in the DNA/rAd5 group. Similarly, Letvin et al. (7) reported that neither DNA/rAd5 regimens nor rAd5 alone regimens expressing SIV Gag, Pol, and Env resulted in significant reductions of set point SIV RNA levels following SIV challenge of Mamu-A*01-negative monkeys, although a transient 1.1 log reduction of peak SIV RNA levels and a survival advantage were observed in a post hoc analysis of all vaccinated groups combined.

One possible explanation for these observations is that the DNA vaccines utilized in these studies were simply not sufficiently potent to enhance protective efficacy in this stringent challenge model. Improved DNA vaccine delivery technologies, such as in vivo electroporation (9, 11) and the use of genetic adjuvants (1, 3), may therefore prove useful in this regard. An alternative possibility is that DNA priming skewed the phenotypes of the vaccine-elicited cellular immune responses. It has been reported that DNA priming prior to rAd5 boosting increased virus-specific CD4⁺ T-lymphocyte responses in both rhesus monkeys (16) and humans (4), and our data are consistent with these observations (Fig. 1B). It is therefore possible that SIV-specific CD4⁺ T-lymphocyte responses that were augmented by DNA priming may have resulted in increased numbers of viral targets for infection (19). Our current data, however, are insufficient to address this possibility conclusively; thus, further studies will be required to address these hypotheses.

The present findings show that a single-shot immunization with rAd5HVR48 vectors afforded a detectable but relatively modest level of protective efficacy against a homologous SIV challenge in rhesus monkeys. Overall, the protective efficacy observed in this study was less impressive than that seen in a previous study in which we utilized a heterologous rAd26 prime, rAd5 boost regimen expressing SIV Gag (10). A direct comparison of the results of these two studies, however, is not possible as a result of the different vectors and inserts utilized. Nevertheless, it appears likely that a single-shot vaccine regimen may not afford as robust protection as an optimal heterologous rAd prime-boost regimen. In our previous study of the rAd26/rAd5 regimen, we observed a significant inverse correlation between the magnitude and breadth of Gag-specific cellular immune responses and set point viral loads (10). In contrast, we did not detect statistically significant correlations between vaccine-elicited immune responses and set point viral loads in the present study. This may reflect the fact that DNA priming could have opposing effects in terms of augmenting potentially useful cellular immune responses but also potentially increasing viral targets for infection, thus complicating analyses of immune correlates of protection.

In the present study, peak and set point SIV RNA levels were lower in animals that received rAd5HVR48 vectors expressing Gag, Pol, Nef, and Env than in those that received rAd5HVR48 vectors expressing only Gag, Pol, and Nef. These data indicate that inclusion of Env resulted in improved control of SIV replication in this study. We suspect that Env-specific cellular immune responses likely contributed to the improved protective efficacy, since Env-specific NAbs against primary isolate SIVmac251 were not observed either prior to challenge or following challenge. More rapid kinetics of Env-specific ADCVI activity, however, was observed in the groups administered vaccine containing Env; thus, it is possible that nonneutralizing Env-specific antibodies also contributed to protection. A limitation is that the challenge virus was essentially homologous to the antigens contained in the vaccine. Thus, future studies utilizing larger numbers of animals will be required to evaluate the potential utility of Env against heterologous SIV challenges. The protective efficacy of single immunizations of optimal vaccine vectors should also continue to be explored.

We thank R. Dolin, N. Letvin, D. Roberts, A. Nanda, P. Swanson, J. Arthur, F. Stephens, and M. Pensiero for generous advice, assistance, and reagents.

We acknowledge support from the National Institutes of Health to D.H.B. (grants AI058727, AI066305, AI066924, and AI078526).

We declare no competing financial interests.