The cytokine interleukin-2 (IL-2) has been studied as a potential HIV therapy for over fifteen years. IL-2 is produced naturally by a number of different immune system cells, including CD4 T cells. IL-2 was originally christened "T cell growth factor" due to its ability to trigger T cells to copy themselves (proliferate). Researchers hypothesized that IL-2 might be able to increase CD4 T cell counts in people with HIV infection, and thus restore immune function and delay disease progression. While several studies have now demonstrated that IL-2 (given either intravenously or subcutaneously) can increase CD4 T cell counts, the impact on immune function has not been thoroughly investigated.
In the following study, researchers from the AIDS Clinical Trials Group (ACTG) analyzed responses to a variety of immunizations in 13 individuals receiving Highly Active Anti-Retroviral Therapy (HAART) and 25 individuals receiving HAART + IL-2. The results showed that despite having higher average CD4 T cell counts (865 vs. 445), people that received IL-2 did not mount better responses to immunization than those receiving HAART alone. In fact, IL-2 recipients tended to have lower proliferative responses to tetanus and inactivated HIV immunizations. Additionally, significantly fewer IL-2 recipients developed antibody responses to hepatitis A vaccination compared to the HAART only group (88% vs. 36%). While seemingly counterintuitive, these results suggest that IL-2 may increase CD4 T cell numbers without necessarily improving T cell function. However, this study does not answer the question of whether adding IL-2 to HAART can improve disease-free survival compared to HAART alone; this question is being addressed by two large ongoing clinical trials called SILCAAT and ESPRIT. Results from these trials should become available in the next few years.1
One of the earliest proposed approaches to treating HIV infection involved the use of therapeutic vaccines. The idea -- most often ascribed to polio vaccine developer Jonas Salk -- was that it might be possible to enhance the immune response to HIV in infected individuals, and thus delay or prevent disease progression. However, results from trials of various candidates (including Salk's whole-killed HIV vaccine, Remune) proved disappointing. The advent of HAART has recently rekindled interest in therapeutic immunization, since it may be easier to induce new immune responses to HIV while drugs are keeping viral replication in check. Novel methods of vaccination that may address some of the shortcomings of previous approaches are also being developed.
In the January issue of Nature Medicine, researchers report the first results obtained with one such novel strategy -- a dendritic cell-based therapeutic vaccine -- in SIV-infected macaque monkeys. Dendritic cells (DCs) are a type of immune system cell that process and present fragments of infectious agents to T cells, a function known as antigen presentation. DCs also provide important "co-stimulatory" signals to responding T cells, and these signals can influence both the magnitude and function of the resulting immune response. Many groups of researchers are now investigating whether the pivotal role played by DCs in inducing T cell responses can be exploited therapeutically. In this study, DCs were isolated from Chinese rhesus macaques and grown in the laboratory. The DCs were then exposed to an inactivated SIV vaccine (leading to the processing and presenting of SIV antigens -- the researchers described these DCs as SIV-loaded) or left unloaded with any antigens.
Ten SIV-infected macaques received a series of injections (at baseline and every two weeks for eight weeks) with the SIV-loaded DCs, and a control group of four SIV-infected macaques received the same series of injections with unloaded DCs. The researchers report that animals given SIV-loaded DCs experienced a 1,000-fold drop in SIV viral load which was maintained out to 42 weeks of follow-up. In contrast, SIV viral load remained unchanged in the control group. In terms of immune responses, macaques immunized with SIV-loaded DCs displayed superior cytotoxic T-lymphocyte (CTL) activity and higher neutralizing antibody titers when compared to controls. These results are impressive and somewhat surprising given that no antiretroviral therapy was employed in the study. In an accompanying commentary, Bruce Walker and Nina Bhardwaj express cautious optimism about the findings, and conclude with the statement: "The striking results of the study are unexpected, and it is imperative to rapidly determine whether similar results can be obtained in other cohorts of primates and whether they can be transferred to humans." A human phase I study of a DC-based vaccine approach is slated to begin enrolling soon through the ACTG (ACTG 5130).2
Atherosclerosis is a condition characterized by a hardening of the arteries. It results from a process in which deposits of cholesterol, cellular waste products, calcium and other substances build up in the inner lining of an artery. There has been concern that protease inhibitor drugs used to treat HIV may accelerate the development of this condition, and a new paper in the Journal of Clinical Investigation (JCI) attempts to address the issue using a mouse model of atherosclerosis. It's important to emphasize that the relevance of these data to humans is still unknown, but the findings of this study provide a basis for further investigations.
The work by Dressman and colleagues began by investigating the effects of protease inhibitors in vitro. Because one of the major features of atherosclerotic lesions is the presence of cells called macrophages containing high levels of fatty substances (lipids), the investigators tested whether the presence of protease inhibitors increased lipid levels in a macrophage cell line called THP-1 or a mixed population of human peripheral blood mononuclear cells (PBMC). They found that, when compared with a control substance, each of the protease inhibitors significantly increased the amount of a fat substance called cholesteryl ester within the cells. The increase in the amount of cell-associated cholesteryl ester associated with each drug seemed to mirror the degree to which they cause elevated blood lipids in humans, with amprenavir (Agenerase) causing the least increase, followed by indinavir (Crixivan) and then ritonavir (Norvir). The researchers next determined whether protease inhibitors affected levels of cell surface proteins known to be involved in the cellular uptake of lipids, and found that levels of one such protein, CD36, were indeed increased. The relative increase in the level of CD36 (with amprenavir, 3.4-fold; indinavir, 6.2-fold; ritonavir, 13.1-fold) also approximated the measured increase in cholesteryl ester accumulation.
These findings suggested that protease inhibitors may be able to promote atherosclerosis independently of their effects on blood levels of cholesterol and triglycerides. To investigate this possibility further, a mouse model of atherosclerosis was employed. This model involves mice that have been bred to lack the cellular receptor for low density lipoproteins (LDL), making them prone to the development of atherosclerosis when fed a normal diet. In this study, a dose of protease inhibitors was selected that did not increase cholesterol and triglyceride levels in the mice, and the animals were also fed a chow diet that minimizes diet-induced atherosclerosis. Analysis of peritoneal (an area of the gut) macrophages revealed that all three protease inhibitors increased the levels of cell-associated cholesterol and/or cholesteryl ester compared to control-treated mice. The levels of CD36 protein in macrophages were also measured, revealing a similar magnitude of increase to that seen in vitro (with amprenavir, 3.1-fold; indinavir, 5.3-fold; ritonavir, 12.9-fold). This effect appeared to be specific for macrophages, as no such increase in CD36 levels was seen in cardiac myocytes, adipocytes, or platelets.
The development of atherosclerotic lesions in the mice was consistent with the degree of cholesterol/cholesteryl ester accumulation in macrophages and the increase in CD36 levels. At the end of the study, ascending and descending aortas were removed and opened, and the areas covered by lesions quantified by image analysis. All of the mice treated with protease inhibitors had significantly greater lesion area than did control animals; those treated with amprenavir had the smallest increase in lesion area and those treated with ritonavir had the largest increase in lesion area. In addition, higher doses of protease inhibitors caused an additional increase in lesion area when compared to lower doses. The role of CD36 was further confirmed by treating mice genetically lacking CD36 with ritonavir. These mice did not develop atherosclerotic lesions. The authors conclude by stressing that further studies will be required to ascertain the true relevance of these results to humans, but nevertheless caution that protease inhibitors may be able to promote atherosclerosis even when blood cholesterol and triglyceride levels appear normal. They suggest investigating the possibility of monitoring CD36 levels in individuals taking protease inhibitors as a potential aid to assessing the risk of developing atherosclerosis.3
Perhaps the greatest paradox of HIV pathogenesis is that the virus causes persistent immune activation and yet also eventually causes immune deficiency. Immune activation represents an ongoing response by many cells of the immune system, particularly CD4 and CD8 T cells, and is characterized by increased expression of cell surface activation markers (CD38 and HLA-DR are the most well described) and increased T cell proliferation. Recent studies have confirmed that the magnitude of immune activation in people with HIV is a strong predictor of the speed of disease progression, independent of HIV viral load levels (the role of immune activation in disease progression was first uncovered in 1989 by the late Janis Giorgi, a highly respected immunologist from UCLA).
A report in the January issue of Nature Immunology by a group of Dutch researchers may represent a breakthrough in the effort to understand how immune activation is linked to the development of AIDS. While investigating the role of particular co-stimulatory molecules (see article on the therapeutic DC vaccine, above) in the immune system, the researchers discovered that mice genetically engineered to overexpress one such molecule (CD27) on their B cells experienced a combination of persistent immune activation and eventual immunodeficiency that closely resembles HIV pathogenesis. Both humans and mice possess two large pools of T cells: naïve cells (which mount responses to newly encountered pathogens, leaving a legacy of memory cells that handle any subsequent encounters with that same pathogen) and memory cells (that have been generated over time from naïve cells by exposure to various pathogens). One of the known effects of HIV infection is a slow but steady depletion of the naïve T cell pool (both CD4 and CD8 cells), and the extent of this depletion correlates with the progression of disease. The mice used in this study experienced the same phenomenon, seemingly driven by the continuous activation of naïve T cells and accompanying generation of new memory T cells. It appears that the expression of CD27 on B cells led to the activation of naïve T cells in response to antigens that -- without the additional stimulation provided by CD27 signaling -- would otherwise have been ignored. The accelerated draining of the naïve T cell pool was associated with the development of the opportunistic infection Pneumocystis carinii pneumonia (PCP) when the mice reached 6-8 months of age.
In discussing their results, the authors note that the study is consistent with the recent shift in understanding of HIV pathogenesis: "For a long time it was thought that direct or indirect cytopathic (cell-killing) effects of HIV-1 accounted for the abundant immune abnormalities in HIV-1. However, it has since been suggested that chronic immune activation, resulting from the inability of the immune system to control HIV-1 replication, might contribute substantially to, and could be the main determinant in, the erosion of the immune system in HIV-1-infected individuals. Our studies with CD70 Tg (transgenic) mice show that persistent immune activation per se can result in a state of lethal immunodeficiency."
Also on the topic of HIV pathogenesis, Danny Douek and colleagues from the Vaccine Research Center at NIH have just published an excellent and detailed article reviewing the latest data and current state of the field in the Annual Review of Immunology.4
The ability of HIV to persist in an inactive or latent state, despite suppression of viral replication by HAART, has now been well described. The major source of this latent reservoir is resting memory CD4 T cells. However, knowledge is lacking regarding the ability of this reservoir to actively produce new viruses, and the circumstances under which such viral production might occur. A recent study from Anthony Fauci's group at the National Institutes for Health offers fresh insights into these questions. The study authors found that latently infected resting CD4 T cells isolated from individuals with detectable viral load (viremic) possessed distinct features when compared to those isolated from people whose viral load was suppressed (aviremic). The initial observation made by the researchers was that latently infected cells from most viremic individuals spontaneously produced virus in the laboratory, but those from aviremic individuals did not.
To try and better understand the cause of this phenomenon, a new microarray technique was utilized to compare the genes being expressed by latently infected CD4 T cells from the two groups of study participants. These analyses revealed that the cells from viremic individuals had a number of genes that were significantly up-regulated (more active). The authors state that "these results suggest that active viral replication in vivo may lead directly or indirectly to up-regulation of a number of host genes in the resting CD4 T cell compartment, including the latent viral reservoir, which, in turn, may provide enough metabolic energy for completion of viral assembly and release of cell-free HIV-1 in the absence of expression of cell surface activation markers."
For more information on the topic of viral latency, an excellent minireview has recently been published by Robert Siliciano and colleagues in the Journal of Virology.5
The ability of HIV to develop mutations that allow the virus to replicate in the presence of antiretroviral drugs (drug resistance) is well known. Up until now, however, there had been no reports of a phenomenon known as drug-dependent enhancement of HIV replication. In this scenario, the virus develops mutations that cause it to be able to replicate better in the presence of drug than in its absence. In a new paper from a research team in Japan led by Saori Matsuoka-Aizawa, evidence is presented of such mutations developing in response to treatment with nelfinavir. The virus isolated by the researchers had accumulated multiple genetic changes in the protease and gag genes, and these mutations were associated with enhanced viral replication in the presence of nelfinavir in vitro compared to when the drug was absent. Laboratory studies revealed that the mutations in gag appeared to be key determinants of the drug-dependent enhancement of viral replication.
Drug-dependent mutants -- particularly picornaviruses whose replication is dependent upon the presence of capsid-binding drugs -- have been known for some time in other viral systems, but this study represents the first described example in HIV. More work is needed in order to understand whether this is an isolated case or representative of a more widespread phenomenon. The authors suggest that their findings offer some support for the use phenotypic drug resistance assays in the setting of treatment failure, since these assays directly measure the ability of HIV to replicate in the presence or absence of a given drug, and therefore should reveal cases of drug-dependent enhancement of viral replication.6
Additional details regarding this study were presented at the XI International AIDS Conference in Barcelona. The Powerpoint slides (PDF) can be downloaded free of charge.
Bhardwaj N., Walker B.D., Immunotherapy for AIDS virus infections: Cautious optimism for cell-based vaccine. Nat Med 2003 Jan;9(1):13-4.
Dressman J., Kincer J., Matveev S.V., et. al.,HIV protease inhibitors promote atherosclerotic lesion formation independent of dyslipidemia by increasing CD36-dependent cholesteryl ester accumulation in macrophages. J Clin Invest 2003 Feb;111(3):389-97.
Douek D.C., Picker L.J., Koup R.A., T Cell Dynamics in HIV-1 Infection. Annu Rev Immunol 2003 Jan 8; [epub ahead of print]
Persaud D., Zhou Y., Siliciano J.M., et. al., Minireview: Latency in human immunodeficiency virus type 1 infection: no easy answers. J Virol 2003 Feb;77(3):1659-65.