Therapeutic immunization in HIV infection has been explored as a complementary approach to combination antiretroviral therapy (cART) . The aim of this measure has evolved over time from the concept of diminishing the exposure to cART , to the more recently proposed goals of reaching a functional cure or helping to purge the HIV reservoir in combination with cART . To be effective, a therapeutic vaccine should likely elicit and/or boost an antiviral immune response able to control viral replication. In the ideal scenario this vaccine-induced immune response would lower viral replication to undetectable levels, mimicking the situation of the minority of patients who spontaneously control viral replication to undetectable levels (termed elite controllers) . Control of HIV replication by cellular immunity may also facilitate the elimination of latent viral reservoir by killing cells actively replicating HIV, after virus reactivation is induced with antilatency drugs . Progress in this regard is hampered by the fact that the correlates of an effective cellular response against HIV are incompletely understood [6,7]. Studies in elite controllers have provided some clues about what might be the characteristics of an effective CD8+ T-cell response in the context of chronic HIV infection . The polyfunctionality of virus-specific T cells (the ability to simultaneously produce several cytokines) , their cytolytic ability , differentiation stage , low level of exhaustion  as well as cross-reactivity  and selective targeting of certain HIV proteins , have all been associated with a more effective HIV control and may provide some guidance for the design of immunogens and vaccine vectors able to induce an effective antiviral T-cell response.
Among the different immunogens and vaccine vectors used in therapeutic vaccine studies, poxvirus vector-based preparations have been extensively studied . The attenuated modified vaccinia Ankara (MVA) strain has received great attention due to its safety profile and the capacity to induce a high level of gene expression and a strong immune response . Several clinical trials have been conducted using MVA-based immunogens with distinct HIV inserts, in healthy volunteers [17–21] and in HIV-infected patients [22–24]. In those previous studies of therapeutic vaccination with MVA, HIV inserts included Gag , Nef [22,24] or a combination of Gag and some CTL epitopes . However, many studies evaluated only the quantity but not the quality of vaccine-induced T-cell responses and did not identify specifically targeted CD8+ T-cell epitopes [23,24]. We have recently reported on a MVA-based immunogen expressing Env, Gag, Pol and Nef proteins of HIV-1 clade B (MVA-B) which was tested as a prophylactic vaccine in a phase-I trial in healthy volunteers. This vaccine candidate was safe, well tolerated  and triggered robust, polyfunctional and durable T-cell responses in the majority of healthy volunteers . Now, we have reported that vaccination with this MVA-B vaccine in HIV-1-infected individuals on cART was safe, well tolerated and immunogenic. However, individuals showed rebound in their virus load above the detection limit upon cART interruption . Thus, the vaccine-induced response may lack crucial characteristics that have been associated with viral control. Although T-cell responses are routinely measured following vaccinations, measurement of T-cell functionality and exhaustion is omitted most of the time following vaccination protocols, losing valuable information. In the present study, we have extended the characterization of vaccine-elicited T-cell responses to investigate whether the functional characteristics of HIV-specific CD8+ T-cell responses stimulated by this MVA-B vaccine, and the level of exhaustion of these cells, may explain the lack of efficiency of MVA-B vaccine-induced responses to effectively control the virus.
Patients and methods
Patients and samples
This study was performed in a subset of patients included in a double-blind, randomized, phase-I clinical trial (RISVAC03) that has recently been reported . The study participants were chronically HIV-1-infected patients on successful viral suppression with cART, and were randomized to receive three intramuscular injections of MVA-B vaccine (20 patients) or placebo (10 patients) at weeks 0, 4 and 16. An analytical treatment interruption (ATI) was performed at week 24 (8 weeks after the last dose of MVA-B vaccine) and the dynamics of viral rebound were assessed during the first 12 weeks after cART interruption. Patients were followed up until week 48 . cART was resumed when national guidelines criteria for the initiation of therapy were reached .
The present immunological substudy was performed in 21 participants (13 vaccines and eight placebos) from whom sufficient cellular samples were available. Cryopreserved peripheral blood mononuclear cells (PBMCs) from baseline, weeks 18, 24 and 36 (12 weeks after ATI) were assayed in this study. Viability of thawed PBMCs samples was always greater than 85%.
Optimally defined, human leucocyte antigen (HLA) class I restricted CD8+ T-cell epitopes were used in the stimulation assays. A detailed list of all optimal peptides used in the assays is given as a table in Supplemental Digital Content 1 https://links.lww.com/QAD/A831. For each participant, a subset of optimal epitopes was evaluated, based on a previous screening of responses by ELISPOT assay at several timepoints during the follow-up period (weeks 0, 6, 18 and 24). All peptides were dissolved in dimethylsulfoxide (DMSO) and tested at a final concentration in the cultures of 2 μg/ml.
Stimulation assays and flow cytometry
All stimulation assays were performed with cryopreserved PBMCs. A detailed description of stimulation and staining conditions is given as text in Supplemental Digital Content 2 https://links.lww.com/QAD/A831, and a complete list of monoclonal antibodies and fluorochromes used in the study appears in Supplemental Digital Content 3 https://links.lww.com/QAD/A831.
Figure 1 shows a representative example of data analysis with the strategy of gating. An initial gating was applied using forward (FSC) and side (SSC) scatter, and then FSC area vs. FSC height to select single cells. Staining with Live/dead was used to exclude dead cells from further analysis. From the population of single live lymphocytes, a gate was placed to select CD3+CD8+ T cells. The simultaneous production of macrophage inflammatory protein 1 beta (MIP1β), interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin 2 (IL2) by CD8+ T cells was analyzed first by setting a gate for each respective cytokine, and then using a Boolean gate platform to generate the complete array of the 15 possible combinations (functional cell subsets) of the four cytokines studied. Figure 1 also shows examples of perforin and granzyme B expression as well as expression of exhaustion markers PD1 and Tim3 by HIV-specific CD8+ T cells.
Level of total response against each particular peptide was calculated as the sum of the positive responses from each of the above mentioned functional cell subsets. The functional profile of each peptide-specific response was analyzed by calculating the contribution toward the total response of: each of the 15 unique functional cell subsets; subsets producing 1, 2, 3 or 4 different cytokines; subsets producing one particular cytokine (IFNγ+ subsets, MIP1β+ subsets, TNFα+ subsets, and IL2+ subsets).
For each peptide, the functional subset with the highest level of response (and thus the subset contributing most to the total response) was selected to further analyze the lytic ability (measuring the content of granzyme B and perforin), and the level of exhaustion (measuring PD1 and Tim3 expression) of HIV-specific CD8+ T cells.
The main characteristics of the study population, and the different parameters evaluated are expressed as median (interquartile range). Comparisons between groups were done using Mann–Whitney U-test and between different timepoints in the same group of patients using Wilcoxon test. Correlations between quantitative parameters were explored using Spearman's rho test, and between qualitative variables using the χ2 test or Fishers's exact test as appropriate. All statistical analyses were performed using the SPSS software version 15 (SPSS Inc., Chicago, Illinois USA). All P values were two-tailed, and were considered as significant only when lower than 0.05.
The main characteristics of participants at the time of inclusion in the study as well as the level of plasma HIV-RNA at week 36 (12 weeks after ATI) are shown in Table 1. There were no significant differences between the vaccine and placebo arms in terms of age, sex, CD4+ cell counts, nadir CD4+ cell counts, acme (precART) HIV-RNA load and distribution of risk factors for HIV infection.
Distribution of HIV peptides used in the assays
A total of 87 different optimal HIV-1 peptides were used in the assays, all defined in terms of HLA restriction and optimal length (Los Alamos National Laboratories HIV Immunology Database) (Supplemental Digital Content 1 https://links.lww.com/QAD/A831). Of them, 61 (70%) were peptides with a sequence included in the vaccine insert (IN peptides), and 26 (30%) were peptides with a sequence not included in the vaccine insert (OUT peptides). The majority of OUT peptides were from regulatory proteins other than Nef (10/26, 38%) and from Env (9/26, 35%). The IN peptides were mostly derived from the HIV Gag protein (28/61, 46%), followed by Pol (16/61, 26%), Nef (13/61, 21%) and Env (4/61, 7%). There was no significant difference between the vaccine and placebo arms in the median number of peptides per patient used in the assays (five [3–8] and eight [4–8] peptides, respectively, P = 0.30). The proportion of peptides used in the assays by protein of origin was: 35, 24, 20, 12 and 10% for Gag, Nef, Pol, Env and Reg proteins with no differences between study arms (P = 0.99). The proportion of IN and OUT peptides used in the assays were 75 and 25%, respectively, with no differences between study arms (P = 0.63).
Level and functional profile of HIV-specific CD8+ response
Before vaccination (baseline, week 0) the median magnitude of the CD8+ response against each individual HIV-1-epitope was similar in vaccine and placebo study arms [0.08% (0.02–0.21) vs. 0.04% (0.02–0.21), respectively, P = 0.36]. Similar results were obtained when this analysis was restricted to epitopes of the same protein of origin (data not shown). Responses to individual epitopes located in the immunogen sequence were then assessed over time, during the vaccination period and 12 weeks after ATI (week 36) in the vaccine and placebo groups. A moderate but statistically significant increase in the magnitude of response was observed in the vaccine group at week 18 compared with baseline [0.12% (0.03–0.27) vs. 0.08% (0–0.21), respectively, P = 0.02]. This increase was short-lived and disappeared by week 24. Analyzing peptides from different proteins of origin separately showed that this boost of immune response at week 18 was driven by responses against Gag and Nef epitopes but not epitopes contained in Env or Pol components of the MVA-B immunogen insert (Fig. 2). As an additional proof of a vaccine-induced boost of response, we analyzed the evolution of response towards HIV epitopes not included in the vaccine insert (OUT peptides) and found no significant changes at weeks 18 and 24 neither in the placebo nor in the vaccine groups (Fig. 2). At week 36, a significant increase in the magnitudes of responses compared with baseline was observed in both groups of participants, independently of the peptide origin or of the peptide being IN or OUT (Fig. 2), in line with a stimulation of the total HIV-specific immune response due to an increase in viral replication (Table 1).
We next examined the functional profile of HIV epitope-specific CD8+ T cells, based on their ability to produce different cytokines upon antigen stimulation. At baseline, the functional profiles were similar in both groups with no major differences (Fig. 3). The majority of response was mediated by cells simultaneously producing MIP1β and IFNγ, (contribution of MIP1β+IFNγ+TNFα-IL2– subset) with a slightly higher frequency of such cells in the placebo [100% (61–100)] vs. the vaccine group [75% (15–100), P = 0.04]. Cells simultaneously producing MIP1β, TNFα, and IFNγ were equally present in both groups [contribution of MIP1β+IFNγ+TNFα+IL2– subset: 0% (0–4) vs. 0% (0–23) in placebo and vaccine arms, respectively, P = 0.16]. The contribution of TNFα was very low in both groups, although slightly higher in the vaccine group [contribution of TNFα+ cells: 0% (0–6) vs. 2% (0–31) in placebo and vaccine group, respectively, P = 0.049]. No significant changes in the functional profile of HIV-specific CD8+ response were observed neither in the placebo nor in the vaccine group during the follow-up period (Fig. 3).
Cytotoxic potential and level of exhaustion of HIV-specific CD8+ cells
The expression of cytotoxicity-associated molecules granzyme B (GrzB) and perforin (Perf) was evaluated in HIV-specific CD8+ T cells. Figure 4 shows the profile of HIV-specific CD8+ T cells directed against peptides included in the vaccine insert (IN peptides) in terms of GrzB and Perf expression, at baseline and at weeks 18, 24 and 36 after vaccination. At baseline, this profile was similar in both study arms. Of the three different cell subsets defined by GrzB and Perf expression, the most frequent was GrzB+Perf– subset with very low levels of GrzB+Perf+ and GrzB–Perf+ subsets.
During the vaccination protocol, the level of GrzB+Perf– cell subset significantly increased at week 18 compared with baseline in HIV-specific CD8+ T cells in the vaccine [59% (44–69) and 47% (38–59), respectively, P = 0.001] but not the placebo group, and this increase was maintained at week 24 (P = 0.01) (Fig. 4). Interestingly, this increase in GrzB+Perf– cell subset was not observed in HIV-specific CD8+ cells directed against peptides not included in the vaccine insert (OUT peptides; Supplemental digital content 4 https://links.lww.com/QAD/A832). At week 36, 12 weeks after ATI and rebounding virus, the level of this cell population (GrzB+Perf–) in HIV-specific CD8+ T cells was increased compared with baseline in both study arms (P = 0.003 and P = 0.06 for vaccine and placebo groups, respectively) and this was independent of peptides being IN or OUT. Levels of the other two cell subsets (GrzB+Perf+ and GrzB–Perf+) did not significantly change at any timepoint during the follow-up.
In parallel to cytolytic function and to see whether vaccination would drive cell exhaustion, PD-1 and Tim-3 markers were assessed in HIV-specific CD8+ T cells. Figure 4 shows their level of expression on HIV-specific CD8+ T cells at baseline and at weeks 18, 24 and 36 after vaccination. At baseline, there were no significant differences between the placebo and vaccine arms, with PD1+Tim3– cells dominating the cell population. A similar profile was observed in cytokine-producing CD8+ T cells responding to polyclonal stimulation (data not shown). In patients receiving the vaccine, PD1+Tim3– HIV-specific CD8+ cells significantly increased at week 24 with respect to baseline [14% (5.7–45) and 8% (1.8–33), respectively, P = 0.01], and PD1+Tim3+ HIV-specific CD8+ cells increased at week 18 (P = 0.006). These changes were observed only in CD8+ cells specific for IN peptides.
MVA-based vaccine vectors expressing different HIV antigens have been tested in several clinical trials as candidates for prophylactic HIV vaccines . Also, several phase I clinical trials have proved their safety and immunogenicity as therapeutic vaccines in HIV-infected patients . To our knowledge, the present study is the first analyzing the effect of an MVA-based therapeutic HIV vaccine on the functional characteristics of CD8+ T-cell-mediated HIV-specific immune response, their cytolytic ability, and level of exhaustion in the setting of a double-blind, placebo-controlled, clinical trial. MVA-B vaccine was able to increase the magnitude of preexisting HIV-specific CD8+ T-cell responses, mainly towards Gag and Nef proteins. This boost was, however, transitory and waned 8 weeks after the last dose of vaccination. Before vaccination, HIV-specific CD8+ T-cell response was of limited functionality, with most of the response being mediated by cells producing two of the four cytokines tested, a finding in agreement with previous studies in chronically infected patients . Surprisingly, the functional profile of CD8+ response was not significantly improved by MVA-B vaccination. A few others phase I clinical trials, using MVA-based immunogens as therapeutic vaccines, have reported an increase of CD8+ response  but not others . A more recent trial, performed in a small number of patients receiving an MVA-Nef vaccine but lacking a placebo group, showed no significant variation in the functionality of Nef-specific CD8+ responses but only an increase in magnitude , in agreement with our findings.
Overall, previous studies using MVA vectors in prophylactic HIV vaccines have shown a high degree of immunogenicity [17–20,25], with one of them reporting also an increase in the functionality of T-cell response . This is in contrast to the results in the present study, possibly related to HIV-induced immune suppression in the HIV-infected cohort tested here. In addition, the presence of a preexisting virus-specific response towards immunodominant peptides in the HIV-infected study subjects, may limit the ability of the vaccine to induce new responses and/or improve the existing ones, given that immunodominance epitope patterns are different in vaccine-induced responses than in natural infection . On the other hand, results may vary depending on what type of cellular response is evaluated (CD4+ or CD8+) [22,24], the vaccine regimen and prime-boost strategy used [17,19,31] and more importantly, the read-outs employed to assess immunogenicity and the criteria to define polyfunctionality [17–20,25,26,31].
Given the relevance of the cytolytic markers GrzB and perforin on the cytotoxic function of antiviral CD8+ cells , we also assessed the effect of therapeutic vaccination with MVA-B on their expression. Before vaccination, GrzB and perforin expression by HIV-specific CD8+ cells was similar in both study arms, with a predominance of GrzB expression and considerably lower levels of perforin, as has been previously reported in chronically infected subjects [32–34]. Vaccination was able to slightly but significantly upregulate the expression of GrzB on HIV-specific CD8+ T cells, and this upregulation was maintained 8 weeks after the last dose of vaccine, whereas perforin expression was not affected by the vaccine. As a limitation of our study, we did not perform ex-vivo CTL killing assays to test if this increase in GrzB expression was in parallel to an increase in the ability of CD8+ cells to lyse HIV-infected target cells. However, previous studies have shown a good correlation between the expression of lytic-associated molecules and the ability of HIV-specific CD8+ cells to eliminate infected targets . As has been previously observed with the poxvirus-based ALVAC therapeutic vaccine , a certain level of vaccine-induced activation of viral replication could be responsible for the upregulation of GrzB. However, this effect was observed only on CD8+ T cells specific for HIV peptides included in the vaccine insert, suggesting that it was due to a vaccine-induced stimulation. However, GrzB expression on HIV-specific CD8+ cells was also increased in both study arms at week 36, i.e. 12 weeks after treatment interruption and rebounding viral replication, questioning the antiviral benefits of the vaccine-induced increase of GrzB expression at week 18. Regarding perforin expression, only a single previous study using an MVA-based therapeutic vaccine has reported a small and transient increase in perforin expression . Another study using a dendritic cell-based therapeutic vaccine has shown that vaccine-induced CD8+ cells expressing perforin were directly correlated with viral suppression . Our results do not show an MVA-B vaccine-induced upregulation of perforin expression. The different methodology employed to detect virus-specific CD8+ T cells (tetramer-based assay) in other studies may explain this discrepancy, as cytokine-based and tetramer-based assays may not detect the same subpopulations of virus-specific T cells .
Finally, T-cell exhaustion has been shown to be an important mechanism of CD8+ T-cell dysfunction in the setting of chronic HIV infection . Upregulation of some markers associated with T-cell exhaustion is well documented in chronic HIV infection [12,39,40], and the blockade of the interaction with their ligands has resulted in reversion of functional exhaustion [40,41], emphasizing its therapeutic potential . Therefore, downregulation of T-cell exhaustion would be a desirable effect of any immunotherapeutic strategy aimed to restore T-cell functionality. In the present study, contrary to what was expected, MVA-B induced a significant upregulation of exhaustion markers on HIV-specific CD8+ T cells during the vaccination protocol. This effect was only seen for responses to epitopes contained in the immunogen, indicating that this was a vaccine-mediated effect. However, changes observed at week 36 (when viral load was detectable in both study groups) were observed in both study arms and were independent of peptides being IN or OUT, likely as a consequence of the increase in viral replication, as expression of these markers is correlated with level of viral load [40,43]. As expression of these markers is also directly correlated with T-cell activation , a direct effect of MVA-B on activation of virus-specific cells could be involved in the upregulation of exhaustion markers.
In summary, our results show that although MVA-B vaccine was able to transiently boost HIV-specific CD8+ T-cell response, its effect on several functional characteristics of this response was limited. The upregulation of exhaustion markers on HIV-specific CD8+ T cells during the vaccination protocol could be involved in this scarce performance of the vaccine in terms of CD8+ T-cell functionality, which could explain its limited impact on virological end-points after ATI. Our findings show that it is necessary to evaluate not only the level but the functionality and state of exhaustion of the cells involved in this response, to have full information regarding the effectiveness of a vaccine. Therapeutic vaccination with MVA-B tilts the balance between activation and regulation towards regulation of the response of HIV-specific CD8+ T cells, which impacts on the viral rebound after treatment interruption. Therefore, the search for new poxvirus-based immunogens able to keep an adequate balance between activation and regulation of CD8+ responses is necessary. In this regard, recent preclinical studies using genetically modified poxvirus vectors  or immune modulators as adjuvants , have shown promising results. Further clinical trials are warranted to test the immunogenicity of these new formulations.
We especially thank Eduardo Gil and Paula Martinez for their technical assistance. We acknowledge the Spanish HIV-1 BioBank integrated in the Spanish AIDS Research Network (RIS) and collaborating centers for the clinical samples provided. Special thanks to all the volunteers participating in the phase I clinical trial RISVAC 03 to make this study possible.
Members of the RISVAC-03 Study Group: Instituto de Investigación Sanitaria-Fundación Jiménez Díaz, Madrid, Spain: José M. Benito and Norma Rallón. Irsicaixa AIDS Research Institute-HIVACAT, Hospital Germans Trias i Pujol, Badalona, Spain: Beatriz Mothe, Patricia Cobarsi, Miriam Rosás, María C. Puertas, Jorge Carrillo, Juliá Blanco, Javier Martinez-Picado, Bonaventura Clotet and Christian Brander. Hospital Clinic-HIVACAT, IDIBAPS, University of Barcelona, Spain: Nuria Climent, Montserrat Plana, Carmen Alvarez, Sonsoles Sánchez, Agathe León, Judit Pich, Joan Albert Arnaiz, Lorna Leal, Berta Torres, Constanza Lucero, Alberto C. Guardo, Jose M. Gatell and Felipe García. Hospital Gregorio Marañón, Madrid, Spain: José Luis Jiménez, María Angeles Muñoz-Fernández and Juan Carlos López Bernaldo de Quirós. Centro Nacional de Biotecnología, CSIC, Madrid, Spain: Mariano Esteban, Carmen Elena Gómez, Beatriz Perdiguero, Juan García-Arriaza, Victoria Cepeda and Carlos Oscar Sánchez-Sorzano. Instituto de Salud Carlos III, Madrid, Spain: Nuria Gonzalez, José Alcamí and Laura Jiménez. Hospital Reina Sofía, Córdoba, Spain: José Peña.
Sources of funding: This study was partially supported by grants: EC10-153; TRA-094; project RD12/0017 integrated in the Plan Nacional I+D+I and co-funded by ISCIII-Subdirección General de Evaluación and European Regional Development Fund (ERDF). Norma Rallón is a Miguel Servet investigator from the ISCIII (CP14/00198), Madrid, Spain. Beatriz Mothe is a Joan Rodes investigator from the ISCIII (JR13/00024), Madrid, Spain.
Author contributions: N.R., J.M.B., B.M., C.B. and F.G. conceived and designed the study. N.R. and J.M.B. undertook the statistical analyses. N.R., J.M.B., C.B. and F.G. drafted the manuscript. J.C.L.B.d.Q. and M.P. contributed to the study design and data management. N.R., J.M.B., J.M.L. and M.M. performed all the flow cytometry experiments. M.A.M. and M.E. participated in study analyses and manuscript preparation. All authors reviewed and approved the final version of the manuscript.
Conflicts of interest
The authors have no financial conflict of interest with this work .
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