Butler, Scott L. PhD*; Valdez, Hernan MD†; Westby, Michael PhD*; Perros, Manos PhD‡; June, Carl H. MD§; Jacobson, Jeffrey M. MD‖; Levy, Yves MD¶; Cooper, David A. MD, DSc#; Douek, Daniel MD, PhD**; Lederman, Michael M. MD††; Tebas, Pablo MD‡‡
In the era of highly active antiretroviral therapy (HAART), the majority of HIV-infected patients can control HIV replication appropriately. Most treated patients suppress viral load to undetectable levels and increase to variable degrees CD4 T-cell counts.1–5 Survival has improved dramatically, and HAART has made HIV infection a chronic manageable illness. However, despite years of viral suppression, a substantial proportion of patients demonstrate persistent immune activation, which is associated with disease progression and mortality.6–10 In addition, many patients do not achieve complete CD4 reconstitution (≥500 cells/mm3),4,5,11,12 and this has been linked to a high rate of clinical events (Fig. 1).13–19 This may account for the shortened life expectancy in HIV infection, which remains reduced by an estimated 10 years compared with the general population.15,20,21 Although initiating therapy earlier may result in more complete immune restoration and better outcomes,22,23 many patients still present late, and this is associated with diminished immune restoration and shorter survival.4,24,25 These patients will need additional interventions to address the immune activation and incomplete immune restoration that persist during HAART.
THE IMPACT OF CHRONIC IMMUNE ACTIVATION
Immune Activation and CD4 Cell Depletion
Immune activation begins during the acute phase of infection when there is a profound depletion of CD4 cells, mainly from the gastrointestinal tract, and levels of cytokines and chemokines rise with HIV load. During the chronic stage, viral load is reduced, but immune activation persists. This stage is characterized by increased turnover and altered phenotypes of T cells and B cells26–31; raised levels of proinflammatory cytokines including tumor necrosis factor (TNF), interleukin (IL)-1, and IL-6; elevations in C-reactive protein (CRP) and the coagulation marker D-dimer;32,33 and fibrosis of lymphatic tissue, including extensive collagen deposition in the gut-associated lymphoid tissue (GALT).34–36
Although HAART reduces immune activation, virologically suppressed patients on HAART maintain levels of activated CD4 and CD8 T cells that are significantly higher than those in HIV-negative subjects (Fig. 2), both in peripheral blood and lymph nodes.5,37,38 Similarly, elite controllers—patients who maintain viral load <50 copies per milliliter without antiretroviral therapy—have higher levels of activated T cells than uninfected controls.39 Studies show an inverse correlation between T-cell activation and CD4 restoration,37,39–41 both among HAART-treated patients37 and elite controllers.39 Moreover, both high levels of T-cell activation and low CD4 counts have been correlated with clinical events.6,7,14,17,18 Thus, modulating immune activation may affect CD4 count and vice versa, and it is unclear whether modulating either variable will result in clinical benefit. To date, connections among immune activation, immunodeficiency, and adverse outcomes are based on correlation studies.
Immune Activation and Cardiovascular and Other Clinical Events
Markers of inflammation and coagulation are also elevated in HIV-positive persons compared with uninfected controls.33 A comparison of HIV-infected patients from the Strategies for Management of Antiretroviral Therapy (SMART) study with participants from 2 population-based studies showed that SMART participants had higher levels of IL-6, hsCRP, D-dimer, and cystatin-C, although the majority of SMART participants were virologically suppressed on HAART.33 The increased levels of IL-6 and hsCRP were predictors of clinical events, including all-cause mortality and AIDS-related opportunistic infections.9,10,33,42 Other studies have corroborated the association between elevated hsCRP and cardiovascular events43 or progression of atherosclerosis.44,45 More broadly, indices of activation and inflammation in HAART-treated patients have been associated with clinical events and mortality.9,10,46 Together with associations between low on-treatment CD4 counts and clinical events (Fig. 1),14–19 these findings suggest that persistent immune activation and incomplete CD4 restoration may contribute to the excess risk of morbidity and mortality in HIV-infected individuals, despite suppressive HAART.
POTENTIAL MECHANISMS OF IMMUNE ACTIVATION AND TARGETS FOR INTERVENTION
Drivers of Immune Activation
What drives immune activation? Several hypotheses, not mutually exclusive, have been proposed. Potential drivers include residual HIV replication, microbial translocation, and coinfections. Fibrosis of lymphatic tissue may drive immune activation indirectly by preventing CD4 T-cell restoration. Each of these proposed drivers has implications for intervention.
Residual Viral Replication
If ongoing HIV replication in tissue compartments drives chronic immune activation, suppressing residual replication with intensification of HAART should be beneficial. However, intensification has been attempted with generally disappointing results.47–51 Intensification with abacavir,48 raltegravir,47,49–51 or maraviroc52,53 yielded no consistent evidence of additional viral suppression (as measured by plasma viral load) beyond that achieved by the existing HAART regimen. However, some intensification studies showed reduced T-cell activation47,49,52,53 without effect on residual viremia,52 suggesting that agents such as maraviroc or raltegravir may have direct immunomodulatory effects.
In the small, uncontrolled study of maraviroc intensification by Wilkin et al,52 the decrease in immune activation markers did not correlate with CD4 increases. However, other studies suggest that blocking CCR5 is associated with an enhanced immunologic response.54 Maraviroc has been associated with enhanced CD4 responses compared with other regimens in both treatment-naive and treatment-experienced patients,55,56 independent of viral load suppression.57 The mechanism may be linked to decreased immune activation: in one subanalysis, maraviroc-induced CD4 cell increases correlated with decreases in CD4 and CD8 cell activation.58
In individuals with virologic suppression who intensified their maraviroc therapy for different reasons, the effects on immune activation are controversial. In 2 different single-arm studies,52,59 CD38 expression was diminished with maraviroc, whereas in another recently reported placebo-controlled study, maraviroc was associated with modest increases in CD38 and HLA-DR on circulating and gut mucosal cells.60 The reason for the disparity in results is unclear; however, in the former studies, samples were examined fresh after overnight shipping or in real time, whereas in the latter study, cells were cryopreserved on site and examined in batch at the end of the study.
Damage to the gastrointestinal tract during acute HIV infection is both immunologic (massive depletion of CD4 T cells) and structural. The latter includes damage to the gastrointestinal epithelium with loss of mucosal integrity,61 and may include massive apoptosis of intestinal epithelial cells.62 One cause of this enteropathy could be the loss of Th17 cells, a subset of CD4 T cells known to produce cytokines that help maintain gut enterocytes.63,64 The damaged gastrointestinal tract allows microbial translocation. Consistent with this hypothesis, levels of circulating plasma lipopolysaccharide (LPS),65 bacterial 16S rDNA,66 and peptidoglycan (D. Douek, unpublished data, December 2010) are elevated in HIV-infected patients, and plasma LPS and bacterial DNA levels correlate with the frequency of activated T cells.65
If microbial translocation contributes to immune activation, then repairing the gut epithelium, reducing gut bacterial load, or reducing circulating LPS might dampen it. This is an area of active research. Oral bovine colostrum was shown to promote GALT development and enhance CD4 counts.67 Aryl hydrocarbon receptor agonists promote Th17 cell differentiation in vitro.68 Ongoing trials are evaluating nonabsorbable antibiotics such as rifaximin to decrease gut bacterial load (clinicaltrirals.gov identifier NCT00603616), and the phosphate binder sevelamer has shown LPS-binding properties that could reduce circulating LPS.69
LPS may promote T-cell activation through stimulation of toll-like receptor (TLR) 4.70 TLRs are central to the innate immune system, and exposure of PBMCs to TLR ligands results in CD4 and CD8 T-cell activation.70 This suggests that systemic exposure to TLR agonists may drive T-cell activation and death during the chronic stage of HIV infection. Consistent with this hypothesis, a recent study showed that hydroxychloroquine, which modulates the TLR pathway, reduced immune activation and increased percentages of circulating CD4 cells in HIV-infected immunologic nonresponders.71
Occult or Apparent Coinfections
A large proportion of activated CD8 cells in HIV infection are specific for cytomegalovirus (CMV).72 One study of CMV-seropositive HAART-treated patients showed that valganciclovir suppressed CMV infection and reduced CD8 cell activation, although there was no effect on CD4 cells.73 If immune activation is driven by coinfecting pathogens including CMV, targeting those pathogens may reduce activation. Although valganciclovir failed due to poor tolerability, other agents may prove useful.
Fibrosis of lymphatic tissue is linked to persistent CD4 cell depletion.35,36,74,75 Collagen deposition may disrupt lymph node architecture, preventing maintenance and reestablishment of naive CD4 cell populations.36 Antifibrotic agents such as pirfenidone could prevent fibrosis in the GALT early in infection, limiting the initial massive CD4 cell depletion (for the few patients diagnosed in the acute phase of infection). Using such agents to reverse established fibrosis could help reconstitute CD4 cells. Although to our knowledge there is currently no clinical data on the use of antifibrotics for reversal or prevention GALT fibrosis, a recent study showed that patients with pulmonary fibrosis benefitted from treatment with pirfenidone.76
Mediators of Immune Activation
Proposed interventions may also target immune activation pathways directly. One study is evaluating chloroquine for blocking TLR signaling (clinicaltrials.gov: NCT00819390). Other possibilities include blocking IL-6, IL-1, and TNF, cytokines that contribute to early destruction in the gut.65,77 However, blocking cytokines may have unintended consequences in an already immunosuppressed population: IL-6 and IL-1 help respond to viral pathogens, while blocking TNF is associated with increased mycobacterial infections and lymphomas.78–80
Host Target Cells for the Virus
Because CD4 cell depletion likely contributes to immune activation, other interventions could interrupt the cycle of infection and depletion of host target cells. These interventions would also affect viral replication and could contribute to eradication strategies.
Target Cell Availability
The absence of CCR5 on CD4 T cells renders them uninfectable by R5 tropic HIV.81–83 Targeted gene therapy to downregulate or block CCR5 may confer resistance to HIV-1 acquisition.84,85 Ongoing studies are treating virologically suppressed patients with low CD4 cell counts by removing CD4 cells, genetically modifying them with zinc finger nuclease (ZFN) technology to inactivate CCR5, and reinfusing the cells.85,86 Perez et al86 reported in preclinical studies that adenovirus vector delivery of the CCR5-targeted ZFN pair led to disruption of ∼50% of CCR5 alleles in populations of primary human CD4 T cells and generated HIV-resistant primary CD4 T cells that expanded stably in HIV-infected cultures. When the cells were transplanted into an immunodeficient mouse followed by infection with CCR5-tropic HIV, the ZFN-modified T cells preferentially expanded. This approach is currently being tested in a phase 1 trial at the University of Pennsylvania (clinicaltirals.gov: NCT00842634).
The feasibility of T cell–based and hematopoetic stem cell (HSC)–based gene therapy for HIV-1 has been shown in humans. Mitsuyasu et al87 showed that autologous HSCs transduced with the anti-HIV ribozyme OZ1 was safe and lowered HIV-1 load. Levine et al88 infused patients with autologous CD4 T cells modified by lentiviral vectors to express an antisense to HIV env. In a follow-up study, Tebas et al89 showed a decrease in viral load in the majority (88% [7/8]) of patients after stopping antiretroviral therapy, with one patient maintaining undetectable HIV RNA for 104 days. These novel therapeutic approaches will need to achieve a high bar of viral eradication or functional cure by replicating the outcome in the “Berlin” patient (discussed below)90,91 to proceed to advanced clinical development. Even if such approaches are successful, the challenges for widespread implementation will be considerable.
Target Cell Renewal
Increasing the CD4 count may interrupt disease progression, but will likely need to be coupled with viral suppression or with reducing the infectability of target (CD4) cells to be of lasting clinical value. The cytokines IL-2 and IL-7 are important drivers of proliferation and differentiation of T cells. Although recent results showed no benefit to expanding CD4 T cells using IL-2,92 studies are ongoing with IL-7. In 2 phase 1/2a studies, IL-7 for HAART-treated patients with undetectable HIV RNA and low CD4 counts (100–400 cells/mm3) reconstituted naive and central memory CD4 and CD8 T cells.93,94 Trials will be required to assess the clinical benefit conferred by this result, and it is still unclear what level of evidence must be demonstrated in proof-of-concept studies to justify embarking on large clinical endpoint trials.
PROPOSED INTERVENTIONS AND CLINICAL ENDPOINTS
The results of the IL-2 trials (Subcutaneous Recombinant, Human Interleukin-2 in HIV-Infected Patients with Low CD4+ Counts under Active Antiretroviral Therapy and Evaluation of Subcutaneous Proleukin in a Randomized International Trial) show the challenge of designing a study to demonstrate the clinical benefit of a proposed intervention in the current HAART era. Although IL-2 caused a substantial and sustained increase in CD4 T cells, the increases yielded no benefit in terms of reduced clinical events.92 Unexpectedly, a higher relative risk of clinical progression to AIDS was observed in patients with the greatest expansion of CD4 cells. On further analysis, the expanded CD4 T cells showed characteristics of regulatory T cells.95 A key challenge, then, in developing disease-modifying interventions is showing that a biomarker or combination of biomarkers is a surrogate for disease progression such that influencing that marker translates into clinical benefit before proceeding to large clinical endpoint trials. Current treatment guidelines recommending that nearly all HIV-infected individuals receive antiretroviral therapy make designing trials for new agents even more difficult because the number of events expected to occur in this population is relatively small.96,97
Proof-of-Concept Phase 2 Studies and Phase 3 Trial Design for Potential Therapies
In light of the Subcutaneous Recombinant, Human Interleukin-2 in HIV-Infected Patients with Low CD4+ Counts under Active Antiretroviral Therapy and Evaluation of Subcutaneous Proleukin in a Randomized International Trial results, what level of evidence will give confidence that we can affect the disease state? What are the appropriate markers to use in phase 2 to better predict a positive effect on clinical endpoints in phase 3? In addition to total CD4 counts, markers could include T-cell immunophenotypic analyses (eg, activated and regulatory T cells), responses to immunization, parameters of innate immunity (eg, myeloid and plasmacytoid dendritic cells, NK cells), and changes in the viral reservoir (assessed by a validated assay). Other potential markers include CRP, IL-6, and D-dimer, and for interventions aimed at restoring the gut epithelium, LPS, bacterial 16sDNA, and peptidoglycan. Once we have established reliable markers and demonstrated positive phase 2 results, showing an effect on clinical outcomes will require careful consideration of which patient population might benefit from these interventions.
The target population must be defined in the current treatment context, in which nearly all HIV-infected individuals receive antiretroviral therapy, and the population harboring detectable virus has decreased dramatically. Viremic patients with multidrug resistance have the greatest need for new approaches, but elevated immune activation and declining CD4 counts also make them least likely to respond to immunologic therapies. Moreover, the needs of this population are best addressed by the continued development of new antiretroviral agents with different resistance profiles.
Among patients virologically suppressed on HAART, those who achieve a sustained CD4 count >500 cells per cubic millimeter have a very low incidence of clinical events (Fig. 1),14–18,98 such that demonstrating an effect in this population would require prohibitively large and lengthy studies. Even among HAART-suppressed patients with CD4 <500 cells per cubic millimeter, the low frequency of events would mandate a large study. Individuals initiating HAART with CD4 >200 cells per cubic millimeter also have a low likelihood of immunologic nonresponse4,11,12 and a low risk for future events. Thus, the success of HAART eliminates many patients from the potential population for testing new therapies. However, the substantial number of patients who initiate treatment late (with very low CD4 counts)24,25 may benefit from interventions in addition to HAART. Studies to evaluate such interventions should be designed similarly to those evaluating cancer therapies: if a favorable outcome is not obtained after treatment of a small and prespecified number of patients, the approach should be rejected.
Patients initiating HAART with CD4 counts <200 cells per cubic millimeter may fail to normalize CD4 levels even after years of virologic suppression.4,11,12 These patients also develop clinical events that are concentrated during the first year of HAART, when they are at higher risk of AIDS or death,97 and of non-AIDS events.14,99 This population may benefit from disease-modifying therapies and is targeted in ongoing and planned studies. A current study is investigating whether adding a CCR5 antagonist (maraviroc) to HAART decreases the incidence of immune reconstitution inflammatory syndrome in patients initiating treatment with CD4 counts ≤100 cells per cubic millimeter (clinicaltrials.gov: NCT00988780). A planned study will investigate whether adding a CCR5 antagonist to HAART in late-presenting patients (CD4 counts ≤200 per cubic millimeter or an AIDS-defining event) decreases the incidence of subsequent clinical events, including immune reconstitution inflammatory syndrome and new AIDS-defining and non-AIDS–defining events (clinicaltrials.gov: NCT01348308). Both studies target populations at high risk of events, such that dampening immune activation may show benefit.
IL-7 for patients with suboptimal CD4 responses is also under investigation. A previously mentioned phase 1/2a study showed an expanded nonregulatory CD4 T-cell population with IL-7.93 Planned studies will assess IL-7 in HAART-suppressed patients with CD4 cell counts below the limits of normal, with endpoints including T-cell immunophenotyping and clinical events. A sustained increase in CD4 T cells, associated with some evidence of improved function, could justify a phase 3 trial investigating long-term outcomes. Plasma viral load increases have been observed in some HIV-infected persons receiving IL-7, but the increases are transient and to date of unknown significance.93,94
Eradication and Cure
Preventing or slowing disease progression might be facilitated by eradication of HIV. Strategies aimed at clearing HIV from resting CD4 cells have included the use of histone deacetylase inhibitors in combination with HAART.100,101 Recent data, however, suggests that latent viral reservoirs are established in bone marrow hematopoietic progenitor cells as well, underscoring the need to further understand the scope of HIV reservoirs to effectively target them.102 A 40-year-old HIV-positive patient in Berlin who underwent HSC transplantation from a homozygous CCR5delta32 (CCR5-/CCR5-) donor subsequently demonstrated undetectable virus in the absence of antiretroviral therapy.90,91 Engraftment and division of the CCR5-/CCR5- HSCs may have deprived the virus of target cells, thus the case may be proof-of-concept for the disease-modifying strategy of rendering target cells uninfectable. However, chemotherapy, the lymphodepletion regimen, immunosuppression to prevent graft-versus-host disease, or the transplantation itself (via graft-versus-host disease) may have contributed. To test this hypothesis in HAART-treated patients with lymphoma, one could measure the latent viral reservoir both before and after chemotherapy, then before and after CCR5 inhibition (either pharmacologically or via ZFN technology); this would isolate the impact of blocking CCR5 on the viral reservoir. Finally, although the case of the Berlin patient demonstrates the possibility of rendering target cells uninfectable, it also illustrates the technical challenge inherent in developing a cure for HIV infection because the therapy administered in this case is clinically infeasible for the vast majority of patients.
Chronic HIV infection is associated with persistent immune activation and disease progression. Even patients virologically suppressed on HAART for years demonstrate elevated levels of immune activation and chronic inflammation and may eventually develop clinical events, often non-AIDS–defining events. The cause of chronic immune activation in well-controlled HIV infection is unknown. Although residual viral replication probably plays little or no role, there is increasing evidence that microbial translocation and coinfections may be significant contributors. Potential interventions targeting immune activation are emerging, from approaches aimed at directly interfering with inflammatory pathways to those aimed at preventing microbial translocation or decreasing the availability of infectable target cells. The main challenges to developing such therapies include identifying an adequate target population, choosing acceptable surrogate endpoints, and designing feasible studies that will provide positive proof-of-concept that these interventions will translate into long-term clinical benefit, conferring the confidence necessary to embark on large-scale clinical endpoint trials.
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