Developing a safe and effective HIV vaccine is a global health priority. CD8 T cell immunity is clearly important in controlling viremia in SIV-macaques (2
). Studying how CD8 T cells induce effective immunity and determining the optimal set of HIV-derived CD8+
T cell antigens and utilization of these antigens in appropriate vectors is a major goal of HIV vaccine design.
To understand the breadth of CD8 T cells required to facilitate control of SIV viremia by vaccination, we inserted three immunodominant SIV-specific CTL epitopes, Gag (KP9) and Tat (KSA10 and KVA10), into the influenza virus vector and analyzed the immunogenicity and protective efficacy of the vaccine. In addition, the evolution of immune escape variants of recombinant influenza viruses expressing the three SIV-specific CTL epitopes in pigtail macaques was examined. Despite priming SIV-specific CTL responses to these epitopes, there were no significant differences in the viral loads, loss of peripheral CD4+
T cells or survival between vaccinees and controls indicating that the vaccine did not provide any protection from SIV disease progression. We were unable to confirm that loss of β7high
CD4 T cells in the periphery was an indicator of disease progression as previously reported (45
). The physiological significance of α4β7 in SIV infection of pigtail macaques is unclear and may be minimal.
The inability of the influenza virus vaccine with the three SIV CTL epitopes to control viremia may illustrate the importance of a broader base (>3 epitopes) of CD8 T cell responses in facilitating control of SIV (50
). Alternatively, the three SIV CD8 T cell epitopes that we inserted into the influenza virus may not contain the critical epitopes necessary to control SIV replication in pigtail macaques. Recent studies have illustrated that a narrowly targeted vaccine containing only 3 SIV CTL epitopes inducing SIV-specific CD8+
T cell responses was sufficient to control SIV replication in Mamu-B
Indian rhesus macaques (7
). Future studies in pigtail macaques testing a range of immunodominant epitopes may assist in refining CTL-based control of primate lentiviruses. Furthermore, CD4+
T cell epitopes that generate functionally effective antibodies responses could be incorporated into vaccine vectors to further probe the minimal base of immunity required for protective efficacy.
The faster emergence of immune escape mutants at the Gag KP9 and Tat KVA10 epitopes in the vaccinated macaques identified by both real-time PCR and pyrosequencing ( and ) suggests that the effectiveness of vaccine-induced CTL responses can easily be undermined by rapid viral evolution (52
). This may be the consequence of only a small number of epitopes being targeted or the selection of epitopes that do not effectively control SIV viremia. Mudd et al. recently showed that vaccination with gene fragments containing SIV epitopes more highly associated with control of SIV viremia was more effective in controlling SIV viremia, although this was undermined by immune escape in a small subset of vaccinees (7
). Recently, influenza virus A replicons have been engineered that enable the expression of recombinant proteins and green fluorescent protein (53
). If these vectors could be engineered to express whole HIV or SIV proteins or large gene fragments, they may be able to induce antibody or CD4+
T cell responses.
We initially reasoned that faster CTL escape might reduce virus fitness and lead to some reduction in viremia; however, we did not observe this. The extensive set of sequencing data from pyrosequencing from serial time points of viral RNA extracted from plasma day 10 to week 20 enabled us to identify a putative compensatory mutation (V145A) that was strongly associated with the Gag KP9 K165R escape mutation. This mutation may mitigate the fitness cost associated with the primary CTL escape mutation. Thus, the phenomenon of rapid viral escape and compensation may pose a significant impediment to CTL-based vaccines targeting a small number of epitopes (50
There are several limitations to our studies. First, the CTL responses induced by the vaccines were modest and more potent vaccines may reveal a greater impact on viral transmission, especially at the mucosae, as shown by Mudd et al. (7
). We might also expect, however, that a higher magnitude of CTL response directed to a limited number or particular set of epitopes may drive faster viral escape and compensation as previously demonstrated when immunizing with a recombinant influenza virus construct containing only one immunodominant Gag CTL epitope, KP9 (55
). Second, although there were no significant differences in viral loads between vaccinees and controls, we cannot exclude a modest effect of vaccination. Even compensatory mutations may not fully restore viral fitness. Third, although the putative V145A compensatory mutation was strongly associated with the K165R CTL escape mutation, detailed in vitro
viral competition assays with molecular clones will be required to confirm this.
HIV infections remain a major global health threat, and an effective prophylactic vaccine is urgently needed. Our study demonstrated that a vaccine based on three immunodominant CTL epitopes in pigtail macaques was unable to protect animals from high levels of viremia and CD4 T cell loss after SIVmac251 challenge, despite elicitation of modest SIV-specific responses postvaccination. Rapid evolution of both CTL responses and compensatory mutations are likely to be the major causes of the failure of this vaccine. We conclude that CTL based vaccines need to induce stronger immune responses to specific CTL epitopes that are pertinent to the protection of pigtail macaques from SIV.