We loaded MDDCs with two HIV-1-derived and one non-HIV-1-derived MHC class II binding 13-20mer peptides. Novel induced CD4+ T-cell responses were detected in at least one peptide in 10 out of 12 participants and in 11 of 12 participants 10 and 32 weeks after the first immunization, respectively (Fig. 3a and Table 1). In particular, the non-HIV peptide PADRE induced robust CD4+ T-cell responses in eight out of 12 participants. The two HIV-1-derived MHC II peptides Env570 and Gag298 each induced responses in eight participants. Novel induced helper responses were dominated by CD4+ T cells double positive for TNF-α and IL-2 production, but triple cytokine and single IFN-γ producing responses were also identified (Fig. 3a–c).
Sequencing plasma HIV-1 RNA confirmed that in all individuals the majority of the epitopes or epitope variants previously shown to cross-react in HLA-A*0201 mice  were present before immunization (Table 1). We followed plasma HIV-1 viral load and CD4+ T-cell counts for 1 year prior to and up to 8 months after the dendritic cell immunization (Fig. 4). Initial transient decreased viral loads were observed in five participants (participants 13, 25, 37, 42 and 57). The most pronounced changes were seen in participant 13, who exhibited a 1.08 (4.26–3.18) log10 viral load reduction starting after the first immunization and lasting up to 2 weeks after the last dendritic cell immunization. The CD4+ T-cell counts in this participant were stable but low during the 10 weeks immunization schedule. However, his vaccine induced drop in viral load had rebounded to pre-vaccination levels 163 days after the first immunization, which together with a stable but low CD4 count resulted in HAART initiation. We did not see any overall or sustained change in viral load after immunization (geometric mean log10 3.97 day 0 and log10 3.82 day 58). The CD4+ T-cell counts gradually decreased from mean of 482 cells/μl at day 0 to 434 cells/μl at day 252 (10%), in line with CD4+ T-cell decline over the same period in treatment-naïve individuals.
The immunizations were safe and well tolerated. No allergic or autoimmune reactions were observed. No hematological, hepatic, muscular, pulmonary or renal toxicities were observed by blood testing (data not shown). The most common reaction was mild-to-moderate local irritation at the site of subsequent injections but not at the initial injection. Interestingly, induction of CD4+ T-cell responses did not cause a raise in viral load.
This test-of-concept study is the first to evaluate the safety and immunogenicity of selected subdominant CD8+ T-cell epitopes in treatment-naïve HIV-1-infected individuals. We hypothesized that delivering multiple subdominant CD8+ T-cell epitopes in combination with HIV-1 CD4+ T-helper peptides on potent MDDCs would have the ability to induce new and functional anti-HIV-1 CD8+ T-cell responses in vivo. Using anchor-optimized epitopes, binding to HLA-A0201 and immunogenicity was improved . Targeting conserved epitopes, not targeted during natural infection, may have the potential to reduce plasma viral load and ultimately postpone the onset of HAART or AIDS, as a novel therapeutic strategy.
The natural CTL responses against HIV-1 are known to be important in the control of infection [1–3]. We, therefore, believe that a successful therapeutic vaccination should not reduce or alter already existing CTL responses, but merely add selected specific T-cell responses, hopefully contributing to improved control of the disease. In our study, inducing novel CD8+ and CD4+ T cells responses did not appear to alter the already existing T-cell responses toward dominant CTL or T-helper epitopes targeting HIV-1, CMV, EBV or Flu. Only in two participants responses toward subdominant epitopes already recognized before dendritic cell immunization were boosted. This might be due to the continuously high antigen exposure driving CD8+ T cells to an exhausted state not capable of responding to booster immunizations [34–37]. Exhausted antigen-specific CD8+ T cells lacking functional cytokine responses  may also explain some of the discrepancy between the antigen-specific CD8+ T cells as detected by pentamers versus cytokine production. Indeed, we found that the majority of vaccine-induced IFN-γ and TNF-α producing CD8+ T cells co-expressed high levels of CD57 (data not shown), supporting the theory of exhausted a response with a potential limited life span. Due to material limitations, this labeling was only preformed on a limited number of samples.
The immunization seemed associated with a reduction of viral load in half of the immunized participants already within the first 2 weeks after the first dendritic cell immunization. This limited and transient decrease in viral load may be a sign of insufficient boosting by the cell-based immunization and/or driven by the HIV-1 infection. It is possible that the first immunization generated CTL that targeted the subsequently injected peptide-loaded MDDCs and thereby prevented (or limited) any booster effect. The increasing local reactions observed only after subsequent autologous MDDC immunizations but not after the first could support this finding. Another possibility is that the virus generates escape mutations and thereby avoids the novel induced CTLs . The affect on viral load was most pronounced in participant 13. However, plasma viral load rebounded about 3 months later, resulting in HAART initiation. The fact that this rebound in viral load appeared a relatively long time after the last immunization prompts us to believe that this was not caused by the immunizations.
Although the selected epitopes used in this study were identified within conserved regions of the HIV-1 genome, we do not know the importance of any associated mutations on viral fitness. This strategy of therapeutic vaccination could be further improved by the use of epitopes with associated escape mutations known to have an impact on viral fitness, possibly also restricted to other more relevant HLA types .
We have shown that it is possible to generate new T-cell responses against selected subdominant, but conserved, epitopes in treatment-naive HIV-1-infected individuals despite ongoing HIV-1 antigenic exposure from their chronic HIV-1 infection. This ability to redirect the immune response toward selected relatively immune-silent epitopes is an important proof of concept. Further optimization of immunization strategies could potentially lead to stronger and more durable cellular responses to additional epitopes with the potential to control viral replication better during chronic and/or acute HIV-1 infection. A reduction of viral load would protect individuals against disease progression (prevention of disease) and supplement or avoid the need for harmful and costly antiretroviral drugs. An effective T-cell-based therapeutic vaccine, by lowering viral load, would also minimize the risk of transmission to healthy individuals and thereby provide a prophylactic effect with large impact in high endemic areas.
We are grateful for the expert opinion and the helpful comments and discussions by Philip Goulder and Gregers Gram and critical review by Rebecca Payne. We acknowledge the technical assistance of Birgit Knudsen, Solvej Jensen, Irene Jensen, Anne Jensen, Kirsten Bødker, Phillippa Collins, Dorthe Petersen, Eva Gaardsdal, Charlotte Vajhøj and Heidi Bonde Knudsen. This work was supported by grants from the Danish AIDS Foundation.
Author contributions: A.F. designed research; H.N.K., J.B., M.T., L.V., A.E.P., J.L.H., B.S.A., I.M.S., J.G. and G.K. performed research; H.N.K., I.K. and L.V. analyzed data; H.N.K., I.K. and A.F. wrote the paper.
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