HIV-Associated Immune Activation: From Bench to Bedside

Abstract

HIV infection is associated with a state of chronic, generalized immune activation that has been shown in many studies to be a key predictor of progression to AIDS. Consistent with this model, nonpathogenic SIV infections of natural hosts, such as the sooty mangabeys, are characterized by low levels of immune activation during the chronic phase of infection. The molecular, cellular, and pathophysiological mechanisms underlying the HIV-associated immune activation are complex and still poorly understood. There is, however, growing consensus that both viral and host factors contribute to this phenotype, with emphasis on the role played by the mucosal immune dysfunction (and consequent microbial translocation) as well as the pattern of in vivo-infected CD4⁺ T cells. The observation that antiretroviral therapy (ART)-induced suppression of HIV replication does not fully resolve immune activation provided the rationale for a number of exploratory studies of potential immune modulatory treatments to be used in HIV-infected individuals in addition to standard ART. This review provides an update on the causes and consequences of the HIV-associated immune activation, and a summary of the immune modulatory approaches that are currently under clinical investigation.

Introduction

T he molecular, cellular, and pathophysiological mechanisms by which HIV infection causes AIDS are complex, multifactorial, and still not completely understood. In practical terms, this incomplete understanding of AIDS pathogenesis is the main reason why, despite almost three decades of research, we still do not have an HIV vaccine or a therapeutic strategy that achieves virus eradication. The early hypothesis that AIDS is just the result of uncontrolled replication of a highly cytopathic virus that infects and kills CD4⁺ T lymphocytes until this cellular population is completely and irreversible destroyed is no longer supported by the available scientific evidence. In particular, the observation that simian immunodeficiency virus (SIV) infection of natural host species, such as sooty mangabeys (SM), African green monkeys (AGM), and Mandrills (MND), is nonpathogenic despite high viremia and the short lifespan of infected cells demonstrates that AIDS is not the necessary consequence of any primate lentiviral infections.^1,2 In fact, the current paradigm is that AIDS is the result, in nonadapted (i.e., recently encountered) primate species such as humans and Asian macaques, of an aberrant interaction between the virus and the host immune system. A number of recent studies have identified and characterized several key immunological features of pathogenic HIV and SIV infections that seem to play a crucial role in causing progression to AIDS.

First and foremost, the establishment of a state of chronic, generalized immune activation is a characteristic feature of pathogenic HIV infection in humans and SIV infection in rhesus macaques (RM), the most commonly used primate model of HIV infection, but is absent during nonpathogenic SIV infection of natural host species.^3
–5 The HIV-associated immune activation is typically characterized by a high frequency of immune cell types (i.e., CD4⁺ T cells, CD8⁺ T cells, B cells, NK cells, monocytes, etc.), expressing markers of activation, proliferation, and apoptosis.⁶ In clinical terms, the level of chronic immune activation present in HIV-infected individuals predicts the tempo of progression to AIDS independently from, and more accurately than viral load.⁷ In addition, the efficacy of antiretroviral therapy (ART) in reconstituting the immune system of HIV-infected individuals is strongly correlated with its effect in reducing the prevailing level of immune activation.⁸ Second, pathogenic HIV and SIV infection of humans and macaques, but not the nonpathogenic SIV infections of natural hosts, is consistently associated with a complex dysfunction of the mucosal immune system that is characterized by a loss of integrity of the mucosal barrier in the intestine.⁹ The hallmark of the HIV-associated mucosal immune dysfunction is the translocation of microbial products, such as lipopolysaccharide (LPS) and flagellin, from the intestinal lumen to the systemic circulation. As these molecules are bioactive in vivo, their presence in the systemic and lymphatic circulation contributes to the establishment of high levels of innate and adaptive immune activation.¹⁰ Third, pathogenic HIV and SIV infections are characterized by infection and progressive depletion of the pool of “central memory” CD4⁺ T cells (TCM), a phenomenon that appears to be critical in determining both the irreversible decline of the total CD4⁺ T cell pool (and thus, ultimately, the rapidity of disease progression) and the size of the “stable” reservoir of latently infected CD4⁺ T cell (which is the key obstacle to any HIV eradication strategy).^11,12 The mechanisms responsible for the HIV-associated homeostatic failure of CD4⁺ TCM are complex, and, in addition to direct virus infection, include several effects of the chronic immune activation, such as increased proliferative senescence, disruption of anatomic niches, and excessive bystander apoptosis.¹³

Remarkably, CD4 TCMs from natural SIV hosts appear to be relatively spared from SIV infection both in vivo and in vitro, with this protection being mediated, at least in part, by entry mechanisms involving down-modulation of the expression of the main HIV receptors CD4 and CCR5.¹⁴

Since chronic immune activation is clearly a key player in the pathogenesis of AIDS, it may be useful to include, in the clinical management of HIV-infected individuals, therapeutic interventions aimed at reducing its negative impact on the overall immune function. There are three main reasons why this has not yet happened. First, the idea of treating an immune deficiency with immune suppressive drugs has historically encountered some justifiable philosophical resistance, expressing itself mainly as a patient safety concern. Second, the ability of the current ART regimen to indirectly target the HIV-associated chronic immune activation by reducing virus replication has convinced many in the field that specific immune modulatory strategies may not be necessary after all. Third, and perhaps most important, our limited understanding of the molecular mechanisms responsible for the HIV-associated chronic immune activation has made it impossible to design immunological interventions targeting specific signaling pathways, thus leaving the proponents of this approach with only “generic” immune suppressive drugs such cyclosporine A and mycophenolate.^15
–17 In recent years, however, there has been a growing appreciation that the HIV-associated chronic immune activation is not fully resolved even in the setting of successful ART and that this residual immune activation may be largely responsible for the increased “non-AIDS” morbidity and mortality observed in HIV-infected individuals undergoing long-term ART.^18,19 In this article we discuss our current knowledge of the HIV-associated immune activation, and summarize the immune suppressive approaches that are currently under investigation to limit its extension.

What Causes the HIV-Associated Immune Activation?

A large body of experimental evidence generated in both HIV-infected individuals and SIV-infected RMs indicates that the establishment of a state of chronic, generalized immune activation is a characteristic feature of pathogenic HIV/SIV infection that is consistently associated with disease progression.^3
–5 Typical features of this HIV-associated chronic immune activation include a higher frequencies of T and B cells with an activated phenotype, increased lymphocyte turnover with abnormalities in cell cycle regulation, high levels of activation-induced cell death in lymphoid tissues, high serum levels of proinflammatory cytokines and chemokines, and extensive lymphoid tissue fibrosis. Interestingly, the acute phase of SIV infection in natural hosts is also characterized by a strong innate and adaptive immune response to the virus; in contrast to pathogenic HIV and SIV infections, however, natural SIV hosts, including SM, AGM, and MND, are able to down-regulate these responses in early chronic infection, which remains characterized by low immune activation.^20

–26

The specific mechanism(s) underlying the establishment and/or maintenance of chronic, aberrant immune activation in pathogenic HIV/SIV infections of humans and RM, or its resolution in nonpathogenic SIV infection of natural hosts, are still unclear. However, the prevailing view is that HIV/SIV infections may induce immune activation by multiple, and complex, molecular and cellular mechanisms, with many of these acting synergistically. An obvious cause of immune activation is the presence of virus, which elicits canonical antivirus adaptive immune responses mediated by HIV/SIV-specific T and B cells. In addition, HIV and SIV may directly stimulate the innate immune system via pattern recognition receptors such as the Toll-like receptors (TLR) 7 and 9 expressed on plasmacytoid dendritic cells (pDCs).²⁷ Other virus-related mechanisms of immune activation include the ability of HIV and SIV's gp120 to signal through binding to the CD4 and CCR5 receptors, and potential proinflammatory effects of factors such as Tat and Nef.^28
–30 The role of virus replication in inducing immune activation is demonstrated by the fact that effective ART reduces immune activation in HIV-infected individuals.⁸

However, the viral factor alone cannot explain the HIV-associated immune activation, since natural SIV infections, in which high levels of viral loads are also present, manifest with an attenuated immune phenotype. In this regard, it is interesting that certain functions of Nef, such as the ability to down-modulate the CD3-TCR complex from the surface of infected cells, have been lost during the evolution from SIV to HIV-1. As such, HIV-1 Nef may have acquired a phenotype that favors immune activation since it does not render infected CD4⁺ cells insensitive to restimulation through the T cell receptor.^31,32

A second potential mechanism contributing to HIV-associated chronic immune activation is the mucosal immune dysfunction that is characterized by a profound depletion of CD4⁺ T cells during the early acute infection, and a progressive loss of ability to maintain the intestinal barrier function.^25,33,34 Of note, the HIV/SIV-induced depletion of mucosal CD4⁺ T cells involves preferentially Th17 cells, i.e., CD4⁺ T helper subsets defined by the production of interleukin (IL)-17 and thought to be crucial for the maintenance of mucosal immunity. Depletion of Th17 cells favors a breakdown of the physical and/or biological mucosal barrier that results in the translocation of bioactive microbial products, such as LPS and flagellin, from the intestinal lumen to the systemic circulation.^35

–38 These microbial products may, in turn, cause a broad activation of the immune system by stimulating various immune cell types, namely macrophages and B cells, via their binding to certain TLRs, and triggering “bystander” activation of lymphocytes that are not specific to HIV antigens.³⁹

Consistent with this pathogenetic mechanism, (1) enteropathy with increased apoptosis of enterocytes has been documented in patients with HIV infection and AIDS; and (2) plasma levels of LPS are significantly increased in chronically HIV-infected individuals and SIV-infected RMs, and correlate with the level of systemic immune activation in HIV-infected individuals.^40,41 The presence of a causal link between microbial translocation and immune activation is supported by the observations that (1) natural SIV infections are associated with low immune activation and the absence of microbial translocation, and that (2) in vivo administration of LPS to chronically SIV-infected AGM results in increased immune activation.^{9

–42}

Another factor that can contribute to immune activation throughout the course of HIV infections, and particularly during the later stages of disease, is the presence of environmental pathogens and opportunistic infections. For example, due to the presence of a compromised immune system, latent herpes viruses such as cytomegalovirus and Epstein–Barr virus reactivate more frequently during HIV infection, and may stimulate large numbers of T cells specific for these viral antigens.^43,44 An additional factor that may contribute to the chronic immune activation observed in HIV-infected humans and SIV-infected RM is the perturbation of immune regulatory mechanisms, such as regulatory T cells and/or immune modulatory cytokines (i.e., IL-10).⁴⁵ In addition, chronic HIV-infected individuals and SIV-infected RM appear to be characterized by a vicious cycle of increased production of proinflammatory and proapoptotic molecules, such as tumor necrosis factor (TNF)-α, IL-6, IL-1β, and type I interferons, bystander activation of T cells, and further release of cytokines and chemokines.⁴⁶ In this context, it has been recently proposed that the preferential infection of CD4⁺ TCM, which are anatomically located in lymph nodes and other central lymphoid tissues, may directly favor immune activation by bringing the bulk of the viral antigenic loads in the districts where primary immune responses are generated.¹³

How Does the HIV-Associated Immune Activation Damage the Immune System?

While it is well established that chronic immune activation plays a pathogenic role in the setting of HIV infection, it is still unclear how exactly this immune activation exerts its deleterious role on the host immune system. One mechanism by which generalized immune activation may damage the immune system derives from the peculiar characteristic of HIV to preferentially infect and kill activated CD4⁺ T-helper cells.⁴⁷ Since T-helper cells are a key component to the host immune response, the HIV-associated immune activation, and ensuing virus-mediated killing of activated CD4⁺ T cells, may create substantial “holes” in the repertoire of these cells that will eventually translate in the inability to successfully control a wide range of potential pathogens. In this view, the concomitant presence of high levels of CD4⁺ T cell activation and a virus infecting activated CD4⁺ T cells may result in a vicious cycle of new infection, virus production, and further death of CD4⁺ T cells, a phenomenon even more pronounced for HIV-specific CD4⁺ T cells, which are indeed particularly susceptible to HIV infection.⁴⁸ In addition, the depletion of CD4⁺ T cells (and that of 199 other cell types as well, particularly during the late stages of infection) may trigger a homeostatic response of the immune system that stimulates activation and proliferation of the remaining cells (mainly those with a naive and central memory phenotypes) to replenish the compartment. Unfortunately, this homeostasis-driven CD4⁺ T cell proliferation may provide further targets (i.e., activated cells) to the virus. Importantly, the chronic stimulation of these regenerative compartments that occurs during HIV infection may translate into a premature aging of the immune system.^49,50

An additional key mechanism by which HIV-associated immune activation negatively impacts immune system function is the dysregulation of the architecture of tissues that are crucial for T cell regeneration and function, such as bone marrow, thymus, and lymph node.^13,51 This structural damage of lymphoid tissues translates into ineffective T cell regeneration, further pushing the homeostatic proliferation of the already existing T cells and thus contributing to the pathogenic sequelae described above, including an accelerated immune senescence. Furthermore, in the gastrointestinal tracts of HIV-infected humans and SIV-infected RM high levels of local immune activation are both cause and consequence of the depletion of bulk and Th17 CD4⁺ T cells, loss of mucosal barrier integrity, and microbial translocation.^52

–56 Finally, the HIV-associated immune activation is characterized by the presence of large numbers of activated lymphocytes producing proinflammatory and/or proapoptotic cytokines, thus sustaining the generalized immune activation and promoting the death of bystander CD4⁺ and CD8⁺ T cells.^57,58 In conclusion, the sustained, chronic immune activation established during pathogenic HIV and SIV infections in humans and RM appears to undermine the integrity and functionality of the host immune system utilizing multiple interrelated pathogenic mechanisms. The ability to target these pathways with specific interventions may be important to optimize the treatment of HIV infection in humans.^59,60

Effect of ART on the HIV-Associated Immune Activation

The introduction of potent ART made it possible to achieve a durable control of viral replication and a marked improvement of the immune function in the majority of treated patients.^61

–65 Unfortunately, while ART significantly reduces the morbidity and mortality associated with HIV infection, none of the currently available protocols induces a state in which interruption of therapy is followed by persistent suppression of virus replication (i.e., functional eradication of the infection).^66

–69 In ART-treated HIV-infected individuals with undetectable viremia the level of immune activation is dramatically reduced compared to baseline (i.e., pretreatment) but rarely goes back to normal levels.⁷⁰ This residual immune activation is associated with long-term sequelae of HIV infection such as accelerated atherosclerosis, neurological disease, metabolic disorders, and several other conditions that may ultimately dictate the life expectancy of HIV-infected individuals in the twenty-first century.^39,71 In addition, in ART-treated HIV-infected individuals the levels of immune activation of the lymphoid reservoirs of latently infected cells appears to be a determinant of the level of residual virus replication.⁷² The mechanisms underlying this residual immune activation are complex and may include persistent low levels of virus replication (i.e., in anatomic compartments that are less sensitive to ART) as well as the presence of irreversible immunological defects that affect the normal ability to down-modulate an immune response once the antigen has been removed.^73,74 In this context, an area of intense investigation is how ART may improve the HIV-associated mucosal immune dysfunction, and thus its role as a contributor to HIV-associated chronic immune activation.

Initial reports showed that despite prolonged ART and recovery of CD4⁺ T cell homeostasis in peripheral blood most HIV-infected patients experience only an incomplete restoration of the gastrointestinal CD4⁺ T cells.^75

–78 However, more recent data showed that CD4⁺ T cell reconstitution at the level of the intestinal mucosa can be observed after long-term ART or when therapy is initiated early in the course of HIV infection.^77,79,80 A similar result was also achieved with early initiation of ART in SIV-infected rhesus macaques.⁸¹

The possibility that residual immune activation is related to persistent virus replication suggests that a more potent antiretroviral regimen (i.e., ART “intensification”) may be more effective in reducing the HIV-associated immune activation. In one study intensification was carried out for 10 weeks with ART regimens (atazanavir/ritonavir, lopinavir/ritonavir, efavirenz) consisting of drugs that were not used previously in the enrolled patients. Since the median levels of viremia were not significantly different between the preintensification and postintensification period, it was concluded that the levels of residual virus replication, and thus immune activation, may depend on the size of stable reservoir compartments that are established prior to the initiation of ART.⁸² Another study, however, showed that raltegravir intensification of a fully suppressive ART regimen reduced the size of the reservoirs and decreased the levels of immune activation (measured as fraction of CD8⁺ T cells expressing CD38⁺ and/or HLA-DR).⁸³ Further studies on the effects of ART intensification on the residual immune activation will be needed to ascertain whether a complete recovery of the “ideal” pre-HIV infection immune system function can be achieved through virological interventions alone.

In absence of a conclusive result in this sense, the alternative possibility, i.e., that specific immune-based interventions will be necessary to fully recover the immune system function of HIV-infected individuals cannot be excluded. Another potentially interesting therapeutic concept is that the combination of ART with drugs that will reactivate virus replication in latently infected CD4⁺ T cells will help in reducing the size of the reservoir in vivo. While interesting, this approach is not yet part of standard treatment of HIV infection due to the scarcity of drugs that can be safely used for viral reactivation and our limited knowledge on the factors required for latent virus reactivation.⁸⁴

Treating the HIV-Associated Immune Activation

The fact that during the course of HIV infection the extent of chronic immune activation is linked to systemic CD4⁺ T cell depletion and eventual immune failure provides a strong rationale for treating HIV-infected individuals with immune suppressive drugs, even if this may seem a paradox in the setting of an immune deficiency syndrome. Based on the fact that ART-treated patients often continue to show levels of immune activation higher than those of HIV-uninfected individuals despite long-term control of virus replication, a role for immune-suppressive drugs in the clinical management of HIV-infected individuals is increasingly being considered. Several trials have assessed, or are assessing, the safety and efficacy of immunosuppressive drugs to be used in conjunction with ART (Table 1).^85,86

Table 1.

Drug Effects

Drug	Effects	Reference
Cyclosporin A	Transient Increase of CD4 T cells	Andrieu et al.⁸⁸
	Reduction of T cell activation	Rizzardi et al.⁸⁹
	Any virological and immunological advantages during highly active antiretroviral therapy (HAART)	Markowitz et al.⁹⁰
Glucocorticoids	Reduction of immunoactivation, apoptosis, and increase in CD4 T cells	Andrieu et al.⁹²
	Serious side effects	Andrieu et al.⁹³
Mycophenolic acid	Inconsistent virological and immunological effect	Garcia et al.¹⁰¹
		Millan et al.¹⁰²
Rapamycin	Controll of HIV replication by decreasing the expression of CCR5	Heredia et al.¹⁰⁸
Hydroxyurea	Partial reduction of viral load	Montaner et al.¹¹⁰
	Lymphopenic effect	Rutschmann et al.¹¹¹
	Serious side effects	Lori et al.¹¹⁴
	Hydroxyurea and ddI synergize to control viral replication	Lori et al.¹¹⁵
Tumor necrosis factor (TNF) inhibitors	Improvement of HIV-associated symptoms	Ting et al.¹¹⁹
Chloroquine	Reduction of viral load	Sperber et al.¹²¹
		Savarino et al.¹²⁰
	No side effects	Sperber et al.¹²²
	Absence of resistance	Sperber et al.¹²²

Cyclosporine A

Cyclosporine A (CyA) is a cyclic endecapeptide obtained from the fermentation broth of two fungi, Trichoderma polysporum and Cylindrocarpon lucidum. CyA is a powerful immune-suppressive agent that has been used in recipients of kidney, liver, bone marrow, and pancreas transplants, as well as in the treatment of autoimmune disorders.⁸⁷ Through inhibition of NFAT-dependent gene transactivation, CyA inhibits T cell proliferation, effector functions, and activation-induced cell death, and creates an imbalance in the interplay between helper and suppressor T cells in favor of the latter. In particular, CyA reduces the gene expression of proinflammatory cytokines such as IL-2, IL-4, and TNF-α, and up-regulates the expression of the immune suppressive cytokine tumor growth factor (TGF)-β. To date it is not clear whether CyA acts on one clone of cells responding to a single antigen or if it is a more generalized and nonspecific immune suppression. In 1988 Andrieu et al. conducted a study in which CyA was given to eight AIDS patients for 17–66 days and to 25 asymptomatic HIV-infected patients. A relevant increase of CD4 T cells was observed during the treatment in eight patients, but the number of CD4⁺ T cells returned to pretreatment status after CyA interruption.⁸⁸ Later, Rizzardi et al. tested the safety and the immune-modulatory effects of combined CyA and ART treatment in nine patients with primary infection. CyA was administered throughout the first 8 weeks of therapy, then stopped, and ART was continued alone.

The main immunological and virological parameters were evaluated at 64 weeks postinitiation of treatment. Although no significant differences were found between the CyA plus ART and the ART alone cohorts for (1) the fraction of subjects with HIV-RNA below 50 copies/ml (average of 90% in the CyA plus ART and 86% in the ART alone), and (2) the levels of HIV-DNA or HIV-RNA, CyA-treated patients showed a higher number of CD4⁺ T cells with respect to patients treated with ART alone during the follow-up. Based on this finding, it was concluded that the decrease of T cell activation in the early phases of HIV-1 disease has a beneficial impact on the long course of the infection.⁸⁹ On the basis of these promising results, the AIDS Clinical Trials Group (ACTG) conduced a randomized study (ACTG 5138) on 54 treatment-naive, chronic HIV-infected patients that received ART with or without CyA. Forty-eight patients completed the study and did not show any differences with regard to the levels of proviral DNA or CD4⁺ T cells. Based on the disappointing results of this larger study, it was concluded that cyclosporine does not provide any advantage during highly active antiretroviral therapy (HAART) treatment.⁹⁰

Glucocorticoids

Glucocorticoids (GCs) are a class of steroid hormones that binds to the GS receptor, expressed in virtually all cells of the immune system. GCs are part of the feedback mechanism that turns down the activity of the immune system in the setting of inflammation, and are therefore widely used in clinical practice to treat diseases that are caused by an overactive immune system, such as allergies, asthma, autoimmune diseases, and sepsis, as well as to prevent and treat acute organ transplant rejection.⁹¹ GCs mediate a variety of immunosuppressive activities by up-regulating the expression of antiinflammatory proteins and repressing the expression and intracellular signaling effects of proinflammatory cytokines. Based on these immune-suppressive activities, several clinical studies were performed in which GCs were administered as monotherapy or in combination with antiretroviral therapy to HIV-infected individuals. Of note, in these studies GCs consistently resulted in decreased immune activation and apoptosis of T lymphocytes and increased CD4⁺ T cell counts.^92
–94

In one of these studies (ACTG study 349), prednisone administration in 24 HIV-infected subjects on stable ART and with a CD4⁺ T cell count >200 mm³ for>12 weeks before entry resulted in a >40% increase above baseline of CD4⁺ T cell numbers. While of potential interest, the long-term use of GCs may be severely limited by the occurrence of serious side effects. For this reason, selective glucocorticoid receptor modulators with safer toxicity profiles are currently under investigation, and should be considered for future therapeutic studies in HIV-infected individuals.^95,96

Mycophenolic acid

Mycophenolic acid (MPA) inhibits proliferation of T cells by selectively blocking the synthesis of guanosine nucleotides by competing with the inosine monophosphate dehydrogenase enzyme (IMPDH). MPA and its ester derivative mycophenolic mofetil (MMF) have been successfully used since the 1990s for the prevention of acute allograft rejection.⁹⁷ Two features make MPA of particular interest as an immune modulator in the setting of HIV infection. First, its antiproliferative effects are specific to lymphocytes since these cells are dependent on the de novo synthesis of purine nucleotides.⁹⁸ Second, since type II IMPDH (mainly expressed on activated T cells) is five times more sensitive to MPA than IMPDH (mainly expressed on resting T cells), MPA preferentially targets activated T cells.^98,99 Several studies investigated the effects of MPA/MMF administration in HIV-infected patients. In the first pilot study, eight ART-treated HIV-infected individuals with undetectable viral loads were daily treated with MMF for 24 weeks; the addition of MMF resulted in a substantial decrease in the fraction of CD4⁺ and CD8⁺ T cells expressing the proliferating marker Ki-67.

Unfortunately, the effects of MPA/MMF administration in HIV-infected individuals have been inconsistent in additional clinical studies in which variable drug dosing regimens, ART regimens, and patients belonging to different stages of disease were used.^100

–104 As such, we still do not have a clear, definitive answer on how the combined ART plus MPA/MMF therapy will impact immune activation during HIV infection.

Rapamycin

Rapamycin (RAPA) is a macrocyclic lactone antibiotic with immunosuppressive properties, currently used for prophylaxis of organ rejection in patients undergone renal transplant. RAPA exerts its immunosuppressive function by inhibiting mTor, an intracellular Ser/Thr kinase that plays an important role in regulating T cell activation and proliferation.¹⁰⁵ The RAPA-dependent mTor inhibition prevents cytokine-mediated T cell proliferation and reduces the expression of CCR5 on monocytes and lymphocytes.^106
–108 Due to its double role in inhibiting T cell proliferation and CCR5 expression, RAPA is of potential therapeutic interest in the setting of HIV infection. Studies in vitro show the ability of RAPA to enhance CCR5 antagonists antiviral activity by decreasing the expression of CCR5 on the cell surface. In addition, a recent study reports the first clinical trials of RAPA in six HIV-infected individuals with CD4⁺ T cell count >100/ml and viral load <50 copies/ml who received RAPA monotherapy after liver transplantation (LT). Remarkably, RAPA monotherapy was associated with improved control of HIV and HCV replication in all patients.¹⁰⁹ These results suggest the potential benefit of RAPA combined with CCR5 inhibitors for HIV treatment.¹⁰⁸

Hydroxyurea

Hydroxyurea is a 30-year-old anticancer drug that inhibits ribonucleotide diphosphate reductase, thus depleting the pool of deoxynucleoside triphosphates available for DNA synthesis. This feature provides a rationale for combining hydroxyurea with nucleoside analogs, in particular didanosine (ddI), since it competes with dATP for incorporation into DNA. Based on its mechanism of action, hydroxyurea was considered an ideal candidate for a new immunological approach to HIV therapy, with several small studies and large trials that investigated the role of hydroxyurea in clinical practice. These clinical trials of hydroxyurea combined with other anti-HIV drugs have produced conflicting results.

In some of these studies, adding hydroxyurea to ddI and/or d4T provided noticeable anti-HIV activity. For example, the Canadian HIV Trials Network Protocol 080 investigated the addition of hydroxyurea to ddI in HIV-positive subjects with CD4 counts between 100 and 350 cells/mm³. After 4 weeks of only ddI, patients were treated with ddI plus 500 mg hydroxyurea (administered daily or twice a day). The subjects receiving hydroxyurea twice daily experienced a partial reduction in plasma viremia, without a significant change in CD4⁺ T cell counts, while those receiving hydroxyurea daily had no significant change in either viral load or CD4⁺ T cell counts.¹¹⁰ In another study, the combination of stavudine (d4T) plus ddI with and without hydroxyurea (500 mg twice daily) was studied in 144 HIV-positive patients. After 12 weeks of therapy, 19% of patients receiving all three drugs had an HIV-RNA <20 copies/ml, compared with 8% of those in the d4T plus ddI arm. CD4⁺ T cell counts, but not percentages, increased less in the hydroxyurea-containing arm (28 vs. 107), reflecting the lymphopenic effect of hydroxyurea.¹¹¹ In a similar study, Galpin et al. randomized 42 HIV-positive subjects to one of three treatment arms: hydroxyurea alone, hydroxyurea plus ddI, or hydroxyurea plus ddI plus d4T. At 28 weeks, hydroxyurea monotherapy had no significant effect, while triple therapy yielded significant improvements in HIV-RNA and CD4⁺ T cell percentages.¹¹² However, in a larger clinical trial, hydroxyurea was not associated with any additional benefits when combined with a regimen containing d4T and ddI. Moreover, the study was stopped early because of a higher rate of side effects (e.g., pancreatitis, leukopenia, neutropenia, anemia, and peripheral neuropathy) in study volunteers who combined hydroxyurea with their other drugs.^113,114 Renewed hope for hydroxyurea comes from recent findings reported by Lori et al.¹¹⁵ indicating that decreasing the dose of hydroxyurea not only diminishes its toxicity but also increases its antiviral potency, and that the combination of hydroxyurea and ddI (available as a new class of antiretroviral combinations, namely “Virostatics”) synergizes to control viral replication.

TNF inhibitors

TNF inhibitors include blocking anti-TNF antibodies as well as soluble receptors that act as signaling antagonist. Etanercept is an artificially engineered dimeric fusion protein made from the combination of two naturally occurring soluble human TNF receptors linked to an Fc portion of an IgG1. Functioning as a decoy receptor that binds to TNF, etanercept reduces the effect of naturally present TNF.¹¹⁶ In the United States the FDA has licensed this drug for rheumatoid arthritis, polyarticular juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, and plaque psoriasis. A significant amount of data indicates that HIV infection is associated with excessive production of TNF-α, and that the abnormal high levels of this proinflammatory cytokine may induce several of the immune dysfunctions seen in HIV-infected individuals. These data provide a strong rationale for testing the therapeutic potential of etanercept in disease management of HIV-infected patients.

An FDA approved Phase I clinical trial was conducted to evaluate the safety and efficacy of etanercept in HIV-infected subjects who have virologically failed to respond to standard antiretroviral therapy. Encouragingly, the results showed that etanercept might improve the symptoms of HIV-associated aphthous ulcers, cachexia, dementia, fatigue, and fever, as well as help manage concomitant rheumatic diseases and psoriasis.^117
–119 Approaches aimed at targeting other proinflammatory cytokines (i.e., IL-1 and IL-6) in HIV-infected individuals are currently being considered.

Chloroquine

At clinically achievable concentrations chloroquine inhibits HIV-1 by affecting newly produced viral envelope glycoproteins.¹²⁰ The antiviral activity of hydroxychloroquine has been confirmed in vivo by two clinical trials. In a drug vs. placebo pilot study conducted on 38 naive HIV-positive patients, reduced plasmatic levels of viral RNA were reported in patients who had been administered hydroxychloroquine for 8 weeks. No side effects were reported in relationship to this drug.¹²¹ In a second trial, that included 72 HIV-positive subjects, the effectiveness of hydroxychloroquine in reducing HIV-1 RNA was compared to that of zidovudine (AZT). Hydroxychloroquine significantly reduced the HIV-1 RNA levels in the plasma, although this effect was less dramatic than the one obtained with AZT. However, resistance to hydroxychloroquine was not observed, while AZT developed a number of mutations.¹²²

Conclusions

A large number of studies showed that the establishment of chronic, high levels of immune activation is a hallmark of pathogenic HIV and SIV infections in humans and RM, as well as a key predictor of progression to AIDS. Confirming this link between chronic immune activation and disease progression, nonpathogenic SIV infections of natural hosts are typically associated with low immune activation during the chronic phase of infection. While it is widely accepted that chronic immune activation plays an important role in the pathogenesis of AIDS, and that both viral and host factors contribute to this phenotype, the specific molecular, cellular, and pathophysiological mechanisms causing the establishment and the maintenance of the HIV-associated immune activation remain poorly understood. This lack of knowledge is negatively impacting our ability to target immune activation in HIV-infected individuals in a safe and effective way. As such, the elucidation of the cause of immune activation during HIV infection will dramatically improve the design of selective immune modulatory treatments to be used in addition to standard ART therapies.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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