The distinguished safety record of YF17D, coupled to its ability to induce robust and highly functional cellular immune responses in humans make this live-attenuated virus an attractive vector candidate for HIV vaccines. In support of this notion, recombinant live YF17D-based vaccines against other flaviviruses and unrelated pathogens have shown promise in pre-clinical and clinical studies 
. We have recently engineered the backbone of YF17D to express amino acids 45–269 of SIVmac239 Gag 
. This recombinant virus replicated and induced CD8+
T-cell responses in Indian rhesus macaques. Franco et al.
have also demonstrated that a rYF17D encoding HIV Gag p24 induced balanced CD4+
T-cell responses in BALB/c mice 
. We, therefore, attempted to expand upon these studies by creating seven new rYF17D viruses expressing fragments of the SIVmac239 Gag, Nef, and Vif proteins. We chose these antigens since vaccine-induced T-cell responses to these proteins have been associated with control of viral replication in a recent SIV efficacy trial 
. Additionally, a number of studies have linked Gag-specific T-cell responses to lower chronic phase viral loads in HIV-1-infected patients 
We vaccinated a total of eleven Indian rhesus macaques with the rYF17D vectors encoding fragments of SIVmac239 Gag, Nef, and Vif; seven animals received individual constructs while four macaques received all seven rYF17D/SIV vectors. We detected transient viremia in all animals that received single constructs, but only two macaques that were immunized with the seven rYF17D/SIV vectors had positive viral loads. An evaluation of cellular immunity induced by a single vaccination with these vectors revealed low frequency T-cell responses directed against Vif and Nef, while Gag-specific responses were nearly absent. A potential caveat in this analysis relates to the sensitivity of our IFN-γ ELISPOT assay, which might have been reduced since PBMC from these animals produced high background levels of IFN-γ at days 14 and 17 post vaccination. We have recently described this phenomenon, which occurs after YF17D vaccination of rhesus macaques and appears to be caused by the activation of CD8+
. Since this “spontaneous” production of IFN-γ increased the background of our ELISPOT assays during the first few weeks after vaccination, we likely missed low-frequency SIV-specific T-cell responses induced by the rYF17D/SIV vectors.
It is also possible that the insertion of SIV sequences in the E/NS1 intergenic region attenuated the rYF17D/SIV constructs even further and thus decreased their immunogenicity compared to the parental YF17D vaccine. Along these lines, a rYF17D virus expressing enhanced green fluorescent protein in this same genomic region demonstrated delayed replication kinetics in vitro
and induced significantly lower titers of neutralizing antibodies in mice compared to the parental YF17D virus 
. Furthermore, we have recently evaluated the replication of YF17D and a rYF17D expressing a fragment of SIVmac239 Gag encoding amino acids 45–269 in Indian rhesus macaques using the same qRT-PCR assay employed in this study 
. We found that positive viral loads came up earlier and at greater magnitudes in animals that received the parental vaccine compared to recipients of the recombinant construct. If lower replication fitness is indeed limiting the magnitude of SIV-specific cellular responses generated by the rYF17D/SIV candidates, additional booster doses might improve the immunogenicity of these vaccine viruses.
It is important to address genetic stability during the development of live-attenuated RNA virus vaccines. In this regard, we have tested the genetic integrity of the rYF17D/SIV viruses by serially passaging them in Vero cells (Bonaldo et al., unpublished data). Electrophoretic analysis of RT-PCR amplicons from viral RNA extracted at the 15th passage revealed that four of the seven rYF17D/SIV viruses were stable at this time point. The rYF17D/Nef(45–210) construct yielded a unique gel pattern, containing the amplicon corresponding to the SIV insert and two smaller, less intense fragments. We are currently investigating whether these extra bands are the result of a mixed viral population. We also found that two constructs – rYF17D/Gag(76–123) and rYF17D/Vif(1–110) – lost their inserts at the 10th passage. Although this may explain why we could not detect Gag-specific responses in r04170– the rYF17D/Gag(76–123)-vaccinated macaque, it does not account for the low frequency of SIV-specific T-cell responses induced by the other stable rYF17D constructs. Additionally, rYF17D/Vif(1–110) – one of the genetically unstable viruses – induced detectable Vif-specific cellular responses in r05089 (200 SFC/106 PBMC). Therefore, the rYF17D/SIV vectors were poorly immunogenic even though the majority of these viruses were stable in vitro.
Our next step was to test whether the low frequency T-cell responses to Gag, Nef, and Vif induced by vaccination with the rYF17D/SIV constructs could be boosted by a heterologous virus boost. To do that, we immunized eight animals with rAd5 vectors encoding full-length Gag, Nef, and a fusion of the Vif, Tat, Rev, Vpr, and Vpx proteins. Four of the animals had been primed with all seven rYF17D/SIV vectors, while the other four macaques were SIV naïve and served as controls for primary responses induced by the rAd5 (, ). We found evidence that the rAd5 vaccination boosted SIV-specific cellular responses in animals that had received the mixture of seven rYF17D/SIV vectors, as seen by the robust expansion of Vif-specific T-cells in r05028 and r98010, as well as the high magnitude of Nef-specific T-cells in r04137. On one hand, this is an encouraging finding since it suggests that YF17D – a clinically relevant vector platform for inducing HIV-specific T-cell responses – effectively primed SIV-specific cellular responses and thus is compatible with heterologous prime boost vaccine regimens. On the other hand, the heterogeneity in the magnitude and specificity of the responses that expanded after the rAd5 boost suggests that some rYF17D/SIV constructs were more effective than others at priming SIV-specific T-cells. Furthermore, the immunogenicity of the rYF17D/SIV prime did not predict the expansion of anamnestic responses after the rAd5 boost. Macaque r04137, for instance, had no detectable cellular responses to the SIV antigens following the rYF17D/SIV prime and yet this animal mounted the highest frequency of Vif- and Nef-specific T-cells in the rYF17D/rAd5 group ( and ). Conversely, rYF17D/SIV vaccination elicited positive IFN-γ ELISPOT responses to Nef and Vif in rh2138 but this animal did not develop high levels of T-cell responses to these two SIV antigens at week 1 following the rAd5 boost ( and ). The reasons for this high animal to animal variability are not entirely clear, but these results suggest that a more thorough investigation of the immunogenicity and in vivo replicative capacity of these rYF17D/SIV viruses is warranted, especially when rYF17D/SIV vectors are administered simultaneously.
We also noticed a trend toward broader T-cell responses among rYF17D/rAd5 vaccineees compared to the rAd5 group (median of total number of pools recognized: 10 versus 7.5, respectively). However, this difference did not achieve statistical difference (p
0.25). The low immunogenicity achieved by the rYF17D/SIV vectors during the priming stage and the small sample size of our experimental groups (n
4) likely contributed to the comparable T-cell breadth observed in rYF17D/rAd5 and rAd5 vaccinees. Additionally, the fact that rYF17D/rAd5 vaccinees were primed with rYF17D/SIV vectors encoding fragments of Gag, Nef, and Vif and subsequently boosted with rAd5 expressing full-length (i) Gag, (ii) Nef, and (iii) Vif fused to Tat, Rev, Vpr, and Vpx might have restricted the repertoire of vaccine-induced T-cell responses by favoring the expansion of T-cells targeting dominant epitopes, as suggested by previous studies 
. Thus, a rYF17D/SIV prime followed by a heterologous virus boost regimen encoding the same SIV minigenes might result in broader SIV-specific T-cell responses.
In summary, the goal of the present study was to evaluate the immunogenicity of live-attenuated rYF17D/SIV viruses expressing fragments of SIV Gag, Nef, and Vif in rhesus macaques. We found evidence that these vaccine viruses replicated in vivo
, but they engendered low levels of SIV-specific cellular responses. Boosting with rAd5 vectors resulted in robust expansion of SIV-specific T-cells, particularly those targeting Vif and, to a lesser extent, Nef. These anamnestic responses comprised CD4+
T-cells capable of performing up to four functions after stimulation with synthetic peptides. However, priming with rYF17D/SIV had a limited effect on the breadth of SIV-specific T-cell responses that expanded after the rAd5 boost. It is important to note that these rYF17D/SIV vectors are in their first generation and thus there is still room for improvement. For example, a vaccination regimen comprised of two or three doses of rYF17D/SIV might increase the immunogenicity of these vaccine vectors. In support of this, Santos et al
. reported that revaccination of YF17D-immune human subjects with the parental YF17D strain resulted in a 3-fold increase in the percentage of activated CD8+
T-cells in peripheral blood 
. Additionally, modification of the SIV inserts increased the genetic integrity of rYF17D/Gag(76–123) and rYF17D/Vif(1–110) – the two recombinant viruses that became unstable after 10 passages in vitro
(Bonaldo et al
. unpublished data). We are also testing whether macaques immunized with an improved rYF17D/rAd5 regimen encoding matched SIV minigenes can control viral replication after a pathogenic SIV challenge (Martins et al
., unpublished data). Optimized rYF17D/HIV vectors may, therefore, be useful for inducing cellular immune responses against the AIDS virus.