Despite the benefits of antiretroviral therapy (ART), approaches to limit the need for lifelong ART would be of considerable value. An effective therapeutic HIV vaccine administered to individuals on effective ART that would prevent or delay viral rebound and/or CD4+ T-cell decline after interruption of ART could fulfil this role.
A number of therapeutic HIV vaccine studies have been published to date [1–20]. A few have demonstrated encouraging results, but these have tended to be uncontrolled or nonblinded controlled trials [4,5,11,12,15,19]. A number of other studies have found no benefit from therapeutic vaccination [1,3,8–10,13,17,18]. Very few randomized, placebo-controlled trials in chronic HIV infection have been reported [9,10,15,16,18,20] and although most therapeutic vaccines studied to date have demonstrated safety and a degree of immunogenicity, few convincing data suggest that this approach has a beneficial effect on viral replication or disease progression.
A broad and strong anti-HIV immune response is likely required to control viral replication, and therefore should be considered in the development of a therapeutic vaccine approach. To address these issues, a randomized, placebo-controlled trial of a combined therapeutic vaccine was undertaken. The intervention included ALVAC 1452, a canarypox-based vaccine designed to induce/enhance CD8+ T-cell responses, and Remune, a whole, killed HIV immunogen that has been shown to induce helper T-cell responses. It was hypothesized that therapeutic vaccination with ALVAC in combination with Remune in the setting of effectively treated chronic HIV infection would induce anti-HIV-specific immunity and delay viral rebound after discontinuation of ART.
Participants and methods
CTN173 was a multicentre, randomized, double-blind, placebo-controlled trial. Participants were randomized as 1: 1: 1 into three treatment groups: ALVAC with Remune (group 1), ALVAC with Remune placebo (group 2) and both placebos (group 3). Remune was administered at weeks 0, 12 and 20. ALVAC was administered at weeks 8, 12, 16 and 20. At week 24, participants interrupted ART and were closely monitored through week 48. Ethics approval was obtained at each participating site and participants provided written informed consent.
Eligible participants were adults (>18 years) with HIV infection who were receiving at least three antiretroviral agents with HIV-RNA levels viral load less than 50 copies/ml for more than 2 years.
Those individuals not taking a protease inhibitor were required to change their regimen to a protease inhibitor with two nucleoside reverse transcriptase inhibitors at least 2 weeks before interrupting ART. CD4 criteria at screening included the following factors: absolute number more than 500 cells/μl, CD4+ T-cell nadir higher than 250 cells/μl and CD4: CD8 ratio higher than 0.5. Participants must have had no evidence of hepatitis B or hepatitis C coinfection.
Participants who reached week 24, and maintained a viral load of less than 50 copies/ml, interrupted ART. Protocol criteria for restarting ART after week 24 included the following factors: viral load higher than 30 000 copies/ml on two occasions at least 4 weeks apart and not decreasing, decrease in total CD4 cell counts more than 20% and CD4 percentage more than 5% points or total CD4 cell counts less than 350 cells/μl.
vCP1452 is a preparation of a modified recombinant canarypox virus, ALVAC(2), expressing the gene products of the HIV-1 env and gag genes, and a synthetic polypeptide encompassing the known human cytotoxic T-lymphocyte epitopes from the nef and pol gene products . ALVAC placebo was composed of Tris-HCl buffer, virus stabilizer and freeze-drying media, reconstituted in isotonic saline. Remune is chemically and physically inactivated, gp120-depleted HIV-1, which is processed and highly purified prior to formulation with incomplete Freund's adjuvant (IFA) . Remune placebo was IFA.
CD4 cell counts and viral load (as measured with the Chiron bDNA version 3.0 assay, Emeryville, California, USA) were measured at week −4, 0, 4, 8, 12, 16, 20 and 24. After therapy interruption, viral load measurements were done twice a week for 4 weeks, weekly for 8 weeks and then monthly thereafter or until the participants restarted therapy. CD4 cell counts continued to be done every 4 weeks.
To assess T-helper cell responses, lymphocyte proliferation assays (LPAs) were performed as previously described . Briefly, peripheral blood mononuclear clear cells (PBMCs) were plated in triplicate at 3 × 105 cells per well and stimulated with various antigens. Antigens/mitogens included HIV-1 gag p24 (5 μg/ml; Aventis-Pasteur SA, Marcy L'Etoile, France), whole HIV antigen (component of Remune; 5 μg/ml; Immune Response Corp, Carlsbad, California, USA), pokeweed mitogen (0.1 mg/ml; Sigma, St Louis, Missouri, USA), Candida albicans antigen (10 μg/ml; Greer laboratories, Lenoir, North Carolina, USA) and cytomegalovirus (CMV), CF antigen strain AD169 (1: 100; Biowhittaker, Walkersville, Maryland, USA). Cells were incubated for 6 days at 37 °C prior to pulsing with 3H-thymidine (1 mCi per well) for a further 6 h. Plates were harvested on glass fibre filters and counted in a 1450 microbeta Trilux scintillation counter (Wallac, Boston, Massachusetts, USA). Stimulation index was calculated by dividing the counts per minute in the stimulated wells by the counts per minute from the unstimulated control wells.
To assess CD8 T-cell responses, a modification of a previously described interferon (IFN)-γ/interleukin (IL)-2 dual colour enzyme-linked immunospot (ELISPOT) assays was used . Thawed PBMC isolated at weeks 0 and 24 were stimulated with 15 amino acid peptides with 11 amino acid overlaps corresponding to the HIV-1 clade B sequence (NIH AIDS Research and Reference Reagent Program) organized into pools containing 23–25 peptides corresponding to Gag (5 pools), Pol (10 pools), Nef 2 (pools), accessory (Acc 6 pools) and Env (9 pools) gene products.
Batched PBMC (150 000–200 000 cells) were plated in duplicate into 96-well multiscreen-IP white-walled plates (Millipore, Bedford, Massachusetts, USA) coated overnight with 3 μg/ml each of capture anti-IFN-γ and anti-IL-2 monoclonal antibody (Becton Dickinson/Pharmagen, San Diego, California, USA) and stimulated with peptide pools in which each peptides was at a final concentration of 4 μg/ml for 28 h at 37°C in 5% carbon dioxide. Media alone was used as a negative control; positive controls included anti-CD3 (Research Diagnostics, Flanders, New Jersey, USA) and the CEF peptide pool (32 peptides derived from CMV, Epstein–Barr virus and influenza virus, NIH AIDS Research and Reagent Program). IFN-γ and IL-2 spot forming cells (SFCs) were detected as previously described .
The cut-off for positive single IFN-γ, single IL-2 and dual IFN-γ/IL-2 responses was determined by testing 11 low-risk HIV-uninfected controls with the same peptide stimulation platform. A positive response for IFN-γ, IL-2 and IFN-γ/IL-2 secretion was defined as more than 50 SFCs per 106 PBMCs, more than 31 SFCs per 106 PBMCs and more than 20 SFCs per 106 PBMCs, respectively, and at least three times over the autologous negative control. The magnitude of HIV-specific responses was calculated by obtaining the sum of SFC per 106 PBMC to each peptide pool inducing a positive response after background subtraction. The breadth was defined as the number of peptide pools inducing a response above background. Although more than one peptide per pool could be stimulatory, if the pool stimulated a response above background it was only counted once for whichever functional response it stimulated. Magnitude and breadth calculations were done for each functional subset detected by the dual colour ELISPOT assay.
The primary outcome of this study was time to viral rebound to detectable levels (>50 copies/ml) following ART interruption. A number of other outcomes were evaluated including time to restart ART, time to meet criteria to restart ART, viral load set-point, viral load 12 weeks following ART interruption and safety of both the vaccine and the ART interruption.
Virologic rebound after suppression of HIV replication with ART occurs a mean of 10 days after discontinuing therapy [24,26,27]. With 17 patients in groups 1 and 3, a delay in viral rebound by 10 days can be detected at α = 0.05 with 80% power, assuming a SD of 10. Allowing for a 15% dropout rate, 20 patients were to be recruited per arm.
The effect of treatment on the primary outcome and other survival outcomes were assessed with a log-rank statistic, Kaplan–Meier curves were used to present these data pictorially. Cox proportional hazards models were used to assess the effect of other predictor variables on these times to event. Each of the predictor variables was examined at baseline, at 24 weeks and as the difference between 24 weeks and baseline.
Nonnormal continuous variables, such as magnitude of HIV-specific responses characterized by IFN-γ secretion, were log-transformed prior to analysis to conform to the assumption of normality required by the analysis of variance (ANOVA). Repeated measures ANOVA was used to assess the effects of treatment and time (measured at 0, 12 and 24 weeks) on stimulation index and counts/min.
Analysis of the decrease in CD4 cell counts from the time of interruption of therapy (week 24) until week 48 was carried out using a mixed model with random intercept and slope and carrying forward the values for those who restarted ART.
Between May 2004 and May 2006, 60 participants were screened and 52 randomized to one of the three study arms and were divided as follows: 19 to ALVAC with Remune (group 1), 18 to ALVAC with Remune placebo (group 2) and 15 to both placebos (group 3). Baseline characteristics (age, sex, presence of ‘protective’ human leukocyte antigen (HLA) alleles, CD4 cell counts and viral load prior to initiation of ART) were balanced between arms (Table 1).
Three participants in group 1 and one in group 3 withdrew before week 24. Reasons were withdrawal of consent (n = 2) and noncompliance with ART (n = 2). Therefore, 48 participants who received all vaccinations and maintained viral load less than 50 copies/ml throughout the initial study period interrupted ART at week 24.
Consistent with published trials, all vaccines used in this study were well tolerated. Adverse reactions, and particularly severe adverse reactions, were infrequent and occurred at the same frequency in all study arms. The only exception to this was injection site reactions (ISRs) and the subjective reporting of fever. ISRs, of which all were grade 1 or 2, occurred in seven, nine and three participants in groups 1, 2 and 3, respectively. Subjective fever was reported in five, one and one participants, respectively.
The primary outcome was time to viral load higher than 50 copies/ml (viral load rebound) with the hypothesis that this would be delayed in the group receiving both vaccines compared to both placebos. Median (interquartile range) time to detectable plasma HIV-RNA in the dual vaccine arm was 24.5 (11, 32) days compared with 13.5 (11, 25) days in the placebo arm (P = 0.1964, Wilcoxon rank-sum test). Median time to confirmed viral load higher than 50 copies/ml in the ALVAC alone arm [23.0 (16, 31) days] was similar to that seen in the ALVAC with Remune group (Fig. 1). When the two vaccine groups were combined for analysis, the difference further approached statistical significance [23 (15, 32) vs. 13.5 (11, 25) days, P = 0.097] (Figs 1 and 2).
A virologic parameter of potential significance is the viral set-point. Although often reported as a viral load at a given time point (i.e. 12 weeks after interruption of therapy), a more precise way of quantitating this has been defined. This is the geometric mean of three consecutive weekly viral load values in which the slope between the first two and last two values was between −0.2 and 0.2 log10 copies/ml per week [8,9]. As illustrated in Fig. 1, there was no difference in viral load set-point among the three study groups. Similarly, the mean viral load at week 36 (12 weeks postinterruption) was not different between these three groups and, in fact, for each group was almost identical to the mean viral load prior to initiation of ART (Fig. 1).
Reinitiation of and meeting criteria to reinitiate antiretroviral therapy
The proportion of participants who restarted ART between weeks 24 and 48 (five, three and two participants in each group, respectively) was not different between groups (P = 0.609, Fig. 3a). The reasons for restarting therapy were varied and are outlined in Table 2.
Laboratory criteria for reinitiation of ART were predefined and based primarily on CD4+ T-cell changes. As shown by Kaplan–Meier analysis (Fig. 3b), participants in the dual placebo arm restarted therapy or met CD4 criteria to restart therapy significantly sooner than those who received active vaccine (P = 0.024). By week 36, all participants in the placebo group either restarted therapy or met CD4 criteria to restart therapy, whereas approximately 20% of active vaccine recipients had not met this endpoint by week 48. Again, participants in the two vaccine arms performed in an almost identical fashion.
The decrease in CD4 cell counts from the time of interruption of therapy (week 24) until week 48, carrying forward the values for those that restarted ART, were greater in the placebo group when compared with the ALVAC with Remune arm (P = 0.04), or to the two vaccine arms combined (P = 0.02). Additional sensitivity analyses using observed data after restart or censoring all data after restart showed similar results.
HIV-specific responses characterized by IFN-γ secretion only (both magnitude and breadth) were similar between groups at baseline and did not increase over time in any of the study groups. Between-group responses characterized by the secretion of both IFN-γ and IL-2 were also similar at baseline. Although there was no significant increase in the magnitude of these response in any group, the median breadth of HIV-specific IFN-γ/IL-2 responses increased in those receiving either ALVAC with Remune or ALVAC alone compared with placebo (groups 1 + 2 vs. 3; P = 0.059). In addition, vaccination did not result in an increase in the proportion of individuals that were classified as ‘responders’ to IFN-γ, IL-2 or both.
Lymphoproliferative responses were evaluated at baseline and at weeks 12 and 24. Proliferative responses to non-HIV antigen did not change over time in any study group. In those participants who received Remune along with ALVAC, there was a significant increase in proliferative responses to both p24 antigen and whole HIV antigen at both 12 and 24 weeks. Enhancement in proliferation was documented by increases in both counts per minute (P = 0.0019 for p24 antigen, P = 0.0008 for whole HIV antigen by repeated measures ANOVA) and stimulation index (P = 0.0038 for p24 antigen, P = 0.016 for whole HIV antigen). No change in proliferative responses to either p24 antigen or whole HIV antigen was observed in the groups that did not receive Remune. Proliferative responses in the Remune-containing group were also significantly greater than that observed in the groups that did not receive Remune (P = 0.0558 for counts per minute and P = 0.0452 for stimulation index to whole HIV antigen).
More detailed immunologic and virologic studies were carried out in select participant subsets. There was no significant change in CD4 cell counts, CD4 percentage, CD4/CD8 ratio, expression of HLA-DR or PD-1 on CD4+ T cells or size of the viral reservoir between week 0 and 24 in any of the study arms evaluated (data not shown), suggesting that vaccine-induced changes in these parameters do not explain the vaccine effect observed in the study.
Immune correlates of viral rebound
To determine whether vaccine-induced immune responses could play a role in viral control after stopping therapy, we assessed whether either responses measured at week 24 (immediately before interruption of therapy) or changes in immune responses that occurred from week 0 to 24 correlated with time to viral rebound once therapy was stopped. There were trends observed with respect to an effect of the vaccine-induced IFN-γ and IL-2 responses (i.e. change from baseline to week 24) on the time to viral load rebound for response magnitude (P = 0.058) and breadth (P = 0.095). Such an effect was not observed when vaccine-induced IFN-γ only responses were considered. Similarly, there were no associations between proliferative responses and time to viral rebound.
Virologic correlates of disease progression
The 10–11 day delay in the time to viral load becoming detectable observed in the two vaccine arms did not result in a difference in magnitude of the viral rebound or the steady state viral load. The fact that the two vaccine arms performed in an almost identical fashion with regard to delaying both the time to viral rebound and the time to meet CD4 criteria for reinitiation of therapy suggests that there may be a relationship between these two outcomes. In fact, it was found that in the study population as a whole, the time to viral rebound correlated significantly with the time to restart therapy or meet CD4 criteria (P = 0.0002).
As approximately 20% of participants who received vaccine neither restarted ART nor meet CD4 criteria by week 48 (24 weeks after stopping therapy), data were analysed to determine if failure to restart ART or meet CD4 criteria could be predicted by the time to viral load rebound. As seen in Fig. 3b, only participants who received vaccine failed to meet these endpoints. For vaccine recipients with a time to detectable plasma HIV-RNA of more than 31 days (top quartile), there was a significantly greater likelihood of not restarting ART or meeting CD4 criteria by week 48 (5/9 = 55%) compared with those with a shorter time 31 (3/25 = 12%) days or less to viral rebound (P = 0.017, Fisher's exact test).
In one of the first randomized, double-blind placebo-controlled trials of a therapeutic HIV vaccine administered in chronic infection, we demonstrate that this approach can have a measurable biological effect on viral replication. The likelihood that the delay in viral rebound from 13 days in the placebo group to 24 days in the combined ALVAC with Remune group represents a true delay is supported by the observation that this delay was also observed in the group that received ALVAC alone. Whether or not this brief delay in virologic rebound is clinically relevant is unclear and as no published studies have monitored this as a specific outcome, it is difficult to know if this observation is unique to this intervention.
Despite the delay in viral rebound, neither the viral load 12 weeks after interruption of ART nor the reestablished virologic set-point differed among groups. In addition, this reestablished set-point was virtually identical to the pretherapy viral load, consistent with a recently published study of ALVAC as a therapeutic HIV vaccine in chronic infection .
As illustrated in Table 2, ART was reinitiated for a variety of reasons. In addition to the fact that a number of participants restarted ART for nonprotocol defined reasons, a number of participants declined reinitiation of ART despite meeting the protocol-defined criteria for restarting ART. Ultimately, there was no significant difference between groups in the number of individuals that restarted ART, although the total numbers were small.
Having received vaccine, however, was associated with a significant delay in the composite outcome of restarting ART or meeting protocol defined CD4 criteria to restart therapy (Fig. 3b) and approximately 20% of participants in each vaccine arm failed to meet this endpoint. Using this outcome, rather than only analysing time to meet CD4 criteria, avoids the assumption that individuals who restart ART and never meet CD4 criteria have a favourable outcome. As with time to viral load rebound, the two vaccine arms performed in an identical fashion, suggesting that this did not occur by chance. In addition, although the observed delay in viral load rebound had no apparent effect on the subsequent viral load set-point, the time to viral load rebound after interrupting ART correlated with the time to restart ART or meet criteria to restart therapy. This further demonstrates biologically relevant activity of the vaccine.
To determine if potential control of viral replication or disease progression might be related to specific measures of vaccine-induced anti-HIV immunity, ELISPOT and LPA assays were conducted. Although the induction of HIV-specific cells secreting both IFN-γ and IL-2 was associated with a delay in viral rebound, those cells secreting single IFN-γ alone were not. The potential significance of responses characterized by the secretion of these two cytokines in the development of effective anti-HIV immunity has recently been reported and suggests that the induction of robust IFN-γ and IL-2 responses may be an important characteristic of an effective therapeutic and perhaps prophylactic HIV vaccine [28–31].
As anticipated, helper CD4+ T-cell responses increased in the group that received ALVAC with Remune, but did not change in the groups that received either ALVAC alone or both placebos. The induction of helper CD4+ T-cell responses was not associated with a delay in viral rebound, or with time to restart ART or meet CD4+ T-cell criteria to restart. Favourable outcomes occurred whether or not Remune was included in the vaccine, and therefore whether or not there was induction of CD4 T-cell help. This suggests that induction of CD4 T-cell help, at least to the degree induced here, may not be an important component of an effective therapeutic HIV vaccine.
Although the present results may appear to conflict with those recently reported by Autran et al., the observation that the viral set-point following vaccination and interruption of ART is virtually identical to the viral load prior to initiation of ART is a consistent finding. It is possible that imbalance in the Autran study and differences in pre-ART viral load accounted for both differences in viral load set-points following structured treatment interruption (STI), as well as likelihood of meeting viral load criteria for reinitiation of ART. It appeared to be differences in the occurrence of this endpoint (one viral load value post-STI of more than 50 000 copies/ml) that drove the outcome of that study.
The results observed here also differ from results reported by Kinloch-de Loes et al. from the QUEST study that used the same vaccine protocol, but was carried out in participants treated in the setting of acute HIV infection. Although initially thought to be the ideal disease stage to study a therapeutic vaccine, given the limited extent of immune damage and therefore potential recovery, this concept has not borne out [1–3].
A number of novel and potentially important observations were made in this study. This is the first therapeutic vaccine approach that, in a randomized, placebo-controlled study, has led to a degree of control of viral replication. Although the 10–11 day delay in viral rebound did not translate into a lower viral load set-point and may not be clinically relevant in and of itself, it was associated with a delay in the time to restart ART or meet CD4 criteria to do so. This might serve as an early marker of subsequent CD4 decline and could potentially be used in future studies to limit unnecessary time off ART.
Although these data may not support the adoption of ALVAC or ALVAC with Remune as a therapeutic vaccine approach, approximately 20% of vaccine recipients had what appears to be a clinically meaningful response to vaccination (i.e. neither restarting ART nor meeting CD4 criteria to do so for at least 24 weeks following interruption of therapy), whereas this was not observed in the placebo group. Finding the determinants that explain the favourable response to vaccination of the individuals studied here will provide important insights into the future development of an effective therapeutic HIV vaccine.
This study was supported by operating grants to J.B.A. from the Canadian Institutes of Health Research (CIHR) grant (#FRN 44179) and the Ontario HIV Treatment Network (OHTN) grant (#ROGA103). Support was also provided by the CIHR Canadian HIV Trials Network and CANVAC. Immune Response Corporation provided study materials. Sanofi-Pasteur provided financial support and study materials. J.B.A. is supported by a Career Scientist Award from the OHTN. J.-P.R. and C.T. are supported by the FRSQ Chercher Boursier. R.-P.S. is supported by a Canada Research Chair.
J.B.A. and R.-P.S. have conducted contract research for Sanofi Pasteur and author S.G. is an employee of Sanofi Pasteur. Otherwise, there is no financial and/or personal relationship between any authors and/or other people or organizations that could inappropriately influence their work.
J.B.A., J.-P.R. and C.T. contributed to the study design, participant enrolment and follow-up, data analysis and manuscript preparation. D.A. and R.W. contributed to data analysis. J.S. contributed to the study design, data analysis and manuscript preparation. N.B. contributed to data analysis and manuscript preparation. C.K. and F.S. contributed to participant enrolment and editing of the manuscript. R.-P.S. contributed to study design, data analysis and editing of the manuscript. The authors would also like to acknowledge the following study coordinators who were instrumental in the conduct of this study: Nancy Lamoureux, Helene Preziosi, Genevieve Bujold, Shelley Schmidt and Roverta Halpenny.
This study is registered at Clinicaltrials.gov with the registration #NCT00212888.
This work has been presented in part at the 15th Conference on Retroviruses and Opportunistic Infections, February 2008, Boston, Massachusetts, USA and at the 17th Annual Canadian Conference on HIV/AIDS Research, April 2008, Montreal, Quebec, Canada.
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