In summary, OPAL immunotherapy, either using overlapping Gag SIV peptides or peptides spanning the whole SIV proteome was highly immunogenic and resulted in significantly lower viral loads and a survival benefit compared to unvaccinated controls. The virologic efficacy in OPAL-immunized macaques was durable for 12 months after ART cessation. Our findings on OPAL immunotherapy were observed despite the virulent SIVmac251
-pigtail model studied 
and provide strong proof-of-principle for the promise of this immunotherapy technique.
The OPAL immunotherapy approach is simpler than many other cellular immunotherapies, particularly the use of dendritic cells. The use of DNA, CTLA-4 blockade and viral vector based approaches are also now showing some promise in macaque studies 
, although such approaches have not yet been translated into human studies. This study added peptides to PBMC, however we have shown an even simpler technique, adding peptides to whole blood is also highly immunogenic, a technique that will be more widely applicable (
and unpublished studies).
This is one of the largest therapeutic SIV vaccine studies yet reported. Although it may have been ideal to have studied irrelevant peptide-pulsed autologous cells as an additional control group, we were concerned that this may have magnified the therapeutic effect or obscured any safety concerns. In the end, the vaccination process was both safe and effective.
How well the findings on OPAL immunotherapy translate to humans with acute HIV-1 infection will be determined by clinical trials. Virus-specific CD4 T cells are typically very weak in HIV-infected humans or SIV-infected macaques; dramatic enhancement of these cells were induced by OPAL immunotherapy and this may underlie its efficacy 
. We measured IFNγ-producing T cells in this study since we had not developed polyfunctional ICS assays prior to initiating the study. However, recent cross-sectional polyfunctional ICS assays suggests OPAL immunotherapy can also induce T cells capable of also expressing the cytokines TNFα and IL-2, the chemokine MIP1β and the degranulation marker CD107a (unpublished data).
A ~1.0 log10
reduction in VL would result in a substantial delay in progressive HIV disease in humans and allow a reasonable time period without the requirement to reintroduce ART 
if these findings are confirmed in human trials. Both the control and vaccinated macaques were treated with ART early in this study (3 weeks after infection), which alone can be associated with a transiently improved outcome in humans 
. None-the-less, a massive loss of CD4+ T cells in the gut occurs within 2 weeks of infection 
. Although it may be challenging to identify humans within 3 weeks of infection, this is when HIV-1 subjects typically present with acute infection. The durable control of viremia exhibited by the vaccinated animals is interesting and consistent with other recent macaque studies 
, suggesting the need for re-immunization may not be substantial. We cannot attribute the durable control of viremia to the second set of immunizations; there was only a marginal, non-significant, increase in the difference in VL between OPAL vaccinees and controls before and after the second immunization series. Further studies are required to address the timing and benefit of ART cover during boosting immunizations with OPAL immunotherapy.
Control of viremia was similar for the OPAL-Gag and OPAL-All groups. Gag-specific CD4 and CD8 T-cell responses in OPAL-Gag animals 5.1- and 3.5-fold greater than those in the OPAL-All animals, despite an identical dose of Gag overlapping peptides. This suggests antigenic competition between peptides from Gag and the other SIV proteins. Inducing immunodominant non-Gag T-cell responses by multi-protein HIV vaccines may limit the development of Gag-specific T-cell responses 
. A large human cohort study demonstrated Gag-specific T-cell responses were the most effective in controlling HIV viremia 
. Useful subdominant T cell responses may be particularly susceptible to dominant non-Gag T cell responses 
. The utility, if any, of inducing T-cell responses to non-Gag proteins (i.e. excluding Gag peptides from the vaccine antigens) can be addressed in future studies of this flexible vaccine technology. Therapeutic HIV vaccines may not need to aim for maximally broad multi-protein HIV-specific immunity.
OPAL immunotherapy with Gag peptides is proceeding into initial trials in HIV-infected humans. Additional peptides can readily be added into standard consensus strains mixes to cover common strain or subtype variations between strains with this technology 
. Additional technologies such as toggling variable amino acids peptides may provide further T cell immunogenicity with this general technology 
. Immunotherapy with peptides delivered onto fresh blood may have potential applicability for other chronic viral diseases such as hepatitis C virus infection and some cancers such as melanoma