Important note: Information in this article was accurate in April 2000. The state of the art may have changed since the publication date.
American Foundation for AIDS Research, April 2000
David Gilden
Introduction
IL-2 Boosts Low CD4+ Cell Counts
But the Reservoirs Remain Plugged
Inducing Immune Control of HIV
Combined Strategies
References
A major worry last winter at the 7th Conference on Retroviruses and Opportunistic Infections was the emergence of drug resistance during transient viral breakthroughs while on therapy. In seeming contradiction, many researchers were interested too in promoting HIV immunity by more extended HIV breakthroughs. Such breakthroughs would be allowed during controlled pauses in therapy, or strategic treatment interruptions (STIs). If drug-resistant HIV can appear during the minor blips in viral load that occur by themselves, it should be even more likely to do so during the major viral excursions that occur during STIs.
The concept driving interest in STIs is that the controlled upsurge in HIV that they allow acts like a vaccine, triggering anti-HIV immune responses that could suppress the virus on their own. Then patients could stop treatment permanently even though the drugs do not eradicate the last bit of HIV in their bodies. There ought to be safer strategies for attacking residual HIV than these structured treatment interruptions, ones that do not pose the possibility of mutant HIV that escapes drug control. Indeed, the 7th Conference on Retroviruses and Opportunistic Infections also included many presentations on several "immune-based" therapies (or IBTs) that, like STIs, attempt to marshal the immune system against HIV. The difference between the immune-based therapies and the strategic treatment interruptions is that the IBTs can be used in conjunction with continued drug treatment. This considerably reduces the danger that the HIV will replicate out of control before an effective immune response arises.
Interleukin-2 (IL-2) has been the most widely used IBT. IL-2 is a cytokine, or signaling agent, excreted by CD4+ T-helper cells to stimulate CD8+ cytotoxic T-lymphocytes (CTLs). (CTLs detect and kill virus-infected cells.) It also increases the proliferation and maturation of the CD4+ cells themselves.
But the generalized immune activation triggered by IL-2 has several drawbacks. Almost everyone suffers serious side effects during and after IL-2 therapy, which is generally injected under the skin twice daily for five days every six to eight weeks. Side effects include severe fatigue, headache, diarrhea and other flu-like symptoms. These side effects are dose-related. Persons on the highest doses, around 15 million units per day, can have their dose reduced to relieve the symptoms of IL-2 therapy. Not surprisingly, IL-2 also stimulates HIV production when not administered with concurrent antiretroviral drugs. It is used for this purpose in test-tube HIV cultures.
IL-2 therapy has been tested since the early '90s as a means to counter the depletion of CD4+ cells caused by the infection. This research began in the laboratory of Clifford Lane, M.D., of the National Institute of Allergy and Infectious Disease (NIAID).
"IL-2 is just CD4 lymphocyte growth factor," Dr. Lane has argued."When you give it to people and their CD4 counts go up, they don't get sick." The clinical benefit that Dr. Lane claims remains unproven. IL-2 is currently the subject of two large six-year trials to definitively document to what extent it delays HIV disease progression.
Still, there is no question that IL-2 boosts CD4+ cell production at times when that increase is definitely needed. The French ILSTIM study 1 presented at the Retrovirus Conference administered IL-2 (4.5 million units twice daily for five days every six weeks) to 70 volunteers who could not increase their CD4+ cell counts above 200 despite a year of successful highly active antiretroviral therapy, or HAART. (The trial participants all had on-treatment viral loads below 1,000 copies/mL.) Thirty-four volunteers started IL-2 immediately after entering the trial. The others served as a control group for six months before they too received IL-2. After the initial six-month period, the median CD4+ cell count in the IL-2 group had gone from 144 to 230 cells/mm3. This group's median CD4+ cell count was nearly 500 cells/mm3 at month twelve. By comparison, the control group's median CD4+ count hardly increased at all in the initial period. It went from 132 cells/mm3 to 152 cells/mm3.
Increasing CD4+ cell counts to levels above 200 cells/mm3 is considered crucial because the incidence of AIDS-related opportunistic infections drops off drastically at that point. At least, that is what happens when CD4+ cells go up with successful HAART alone. Again, it is not yet proven that patients have the same degree of functional immune recovery when CD4+ cell counts rise under the influence of IL-2. The French Health Ministry nonetheless acted on the ILSTIM results by establishing an expanded access program for administering IL-2 to patients who meet that trial's inclusion criteria.
The use of IL-2 in persons with low CD4+ cell counts became possible only with the introduction of HAART. Before that, lack of HIV suppression made IL-2 of little use in this population, which exhibited disturbing increases in viral load and no CD4+ cell increases during preliminary IL-2 testing. At the Retrovirus Conference, several presentations proposed a possible alternative to IL-2, namely IL-7,2,3 which regulates the production of new, naïve CD4+ T-helper cells in the thymus gland. As a more specific immune modulator, IL-7 may not raise the immune activation issues that IL-2 does. Rather, it would directly counter thymic insufficiency. (Note, though, that IL-7 is known to promote lymphoma, which people with HIV already are vulnerable to. It also stimulates HIV production, and so could not be used apart from HAART.)
The introduction of HAART suggested a much broader potential use of IL-2: to stimulate CD4+ T-helper cells in blanket fashion so that those cells harboring latent HIV will start producing the virus. The HAART drugs might then block this HIV from infecting other cells while the cells with newly activated HIV quickly die off. In this way, IL-2 could have a purging effect on the hidden reservoir of HIV, enabling HAART to cleanse the body of remaining virus. HIV would be eradicated, and patients could stop therapy without any sign of the virus returning.
Several studies have addressed this possibility. Unfortunately, IL-2's promise has not been confirmed. In one STI trial, Hans Stellbrink 4 and colleagues from across Germany tested a four-drug HAART combination (d4T, 3TC, nelfinavir and saquinavir) with or without IL-2 in 56 treatment-naïve volunteers. The minimum baseline CD4+ count was 350 cells/mm3. The two study arms exhibited the same viral load change, both during treatment and after it was halted. Lymph nodes, checked by biopsy, took somewhat longer to reach undetectable virus levels among those on IL-2 (a mean of 377 days on HAART alone and 422 days on HAART plus IL-2). Administering IL-2 also did not appreciably accelerate the decline in HIV-infected cells. The most dramatic difference was the expected effect on CD4+ cell counts: After one year on treatment, all of those on IL-2 had normal CD4+ cell counts. This was true for only half of those on HAART alone.
Two other studies failed to observe any benefit from adding IL-2 onto HAART before a treatment interruption. NIAID reported on 14 participants who stopped treatment for 26 to 168 days.5 All had viral loads under 500 copies/mL (for two months to three years), and 11 were receiving IL-2 in addition to HAART. Regardless of the IL-2 or how long their HIV had been suppressed, the patients' final viral load during the treatment interruption was close to what it had been before starting HAART.
A group from Barcelona described 12 volunteers undergoing repeated 30-day treatment interruptions every three months. 6 Five of the twelve received two IL-2 cycles (dosed at 3 million units twice daily) before the first interruption. All had had viral loads below 50 copies/mL for more than two years. After two interruptions, IL-2 had made no difference in either viral load rebound or the appearance of a CD4+ T-helper cell response to HIV p24 core antigen.
If IL-2 alone is not enough to activate and purge the latently infected reservoir, perhaps adding additional immune modulators might help. One French study 7 tried combining HAART, IL-2 and interferon-gamma, which should stimulate macrophages in addition to CD4+ T-helper cells. Macrophages can sustain chronic production of small amounts of virus. They form a second major target cell population and reservoir for HIV.
In this case, the experiment was not successful: Ten volunteers without previous treatment received five drugs (AZT, ddI, 3TC, saquinavir and ritonavir) plus the IL-2 and interferon-gamma. By month 18, everyone's viral load had dropped below 20 copies/mL, but HIV-infected cells were still detectable and the virus could be cultured from these cells in all cases. Two study participants who halted treatment at this point experienced rapid viral rebound.
The French results follow a negative experience using HAART and IL-2 plus OKT3. OKT3 is a monoclonal antibody that attaches to the T-cell receptor and together with IL-2 causes rapid T-cell activation – before triggering cell death. In a recently published Dutch study 8 using this technique, three patients were given OKT3 and IL-2 for five days after at least six months of viral loads below 5 copies/mL on a on a six-drug mega-regimen. HIV levels did rise modestly at first in the blood (one person) or lymph nodes (the other two), but there was no evident permanent change in the number of infected cells. Meanwhile, the three patients suffered from massive inflammatory side effects, including kidney failure and seizures in one.
One reason why IL-2, even with added reinforcement, has not performed in attempts to deplete latent HIV stems from the problem of residual replication. Adding immune stimulants might have a measurable effect if HAART eliminated HIV everywhere but in the latent pool of HIV-infected cells. The only source of new virus would be the occasional activation of individual latently infected cells. They would then spew out new virions in a limited cycle of replication dampened immediately by the drugs.
But HAART leaves a certain amount of residual replication occurring in actively infected cells with a rapid turnover. This trickle slowly replenishes the latent HIV reservoir. The latent pool may decline at first, but it reaches a new equilibrium under HAART. Under these conditions, IL-2 probably cannot appreciably alter this new equilibrium. Mathematical modeling presented at the 7th Retrovirus Conference by Alan Perelson and John Mittler at the Los Alamos National Laboratory predicts this lack of success. 9
IL-2 would be more likely to have a significant, durable impact if it promoted the rise of anti-HIV immunity that would detect and eliminate HIV-producing cells. This possibility is not so far-fetched: Since IL-2 causes generalized proliferation of CD4+ T-helper cells, it should boost the number and activity of any cells in this population that react to HIV antigens. Several studies 10,11 have indicated that such CD4+ cells exist, both in untreated patients and those on successful HAART. These cells remain quiescent even as other immune defenses return during successful HAART. IL-2 might be just the thing to switch on these T-helper cells. They could then prompt other immune cells, in particular CD8+ cytotoxic lymphocytes, to mop up the residual HIV infection.
In practice, IL-2 does not seem to have that effect, though the various studies have come to contradictory conclusions. At best, administering IL-2 seems to lead to only a modest increase in CD4+ T-helper cell proliferation when patients' cells are exposed to HIV antigens. 12
One investigation, 13 organized by Kendall Smith, M.D., of Cornell University, utilized volunteers with suppressed HIV during therapy with HAART plus low-dose daily IL-2 (about 2 million units per day). The IL-2 was continued after the antiretroviral drugs were stopped. Among the nine volunteers who stopped, viral loads increased over the first month to a median 348,000 copies/mL. Viral loads then declined at varying rates (which correlated with volunteers' CD8+ cell counts) until they leveled off at a median of 26,000 copies/mL. This final figure may be less than half the volunteers' median viral load before beginning treatment, but it is nonetheless too high for comfort. An additional benefit from the IL-2 is certainly not apparent.
In another study to investigate IL-2's effect on anti-HIV immunity, 14 Swiss researchers selected 34 patients starting on abacavir plus two protease inhibitors. At week 16, they randomized those with viral loads under 400 to either continue without change, add IL-2 (at a dose of about 10 million units twice daily for five days every six weeks), or add the Remune therapeutic vaccine (injected every 12 weeks). Remune consists of killed HIV with its envelope proteins removed. The remaining viral core is coated with a mineral oil-based adjuvant that helps trigger immune cell recognition and response. The participants in this study were subject to intensive immunologic monitoring, including lymph node biopsies at study entry and at week 63.
Nineteen of the participants are now beyond week 63. IL-2 did lead to heightened responses to other disease antigens and to more rapid and profound increases in overall CD4+ cell counts. None of the six on HAART alone showed any increase in immune response to HIV, and only one of the five on IL-2 plus HAART did so. In contrast, five of the seven persons on HAART plus Remune had moderate increases. (ACTG 5057, set to commence this year, will test whether Remune helps prevent treatment failure when added to currently successful HAART therapy. This 96-week trial will study the value of the immune responses provoked by Remune in 352 volunteers.)
Vaccines are not necessarily beneficial. One trial 15 at New York's Aaron Diamond AIDS Research Institute used a vaccine consisting of a canary pox/HIV hybrid virus plus free gp160 (HIV envelope protein) as a therapeutic vaccine in people first treated with HAART within 90 days of developing primary HIV infection. Trial participants had had viral loads below 50 copies/mL for greater than two years. They were taken off therapy completely after a course of four vaccinations over six months. Of the four volunteers described so far, only two developed strong new responses to HIV proteins. These two also displayed slower HIV rebound than the other two. They had viral doubling times of 3.2 and 4.5 days, compared to a doubling time of 1.4 days in the two persons with weaker HIV immune responses.
This study's present small size makes it impossible to make any generalizations from its observations. The degree and durability of protection afforded by the vaccine-induced immunity is subject to the same controversy as that surrounding STI-induced immunity. The persistence of measurable viral load in the two Aaron Diamond volunteers with the best response may presage the eventual collapse of immune control due to the CD4+ cell death triggered by HIV. Vaccines that stimulate a more effective immune defense are in order.
At least six Retrovirus Conference presentations discussed a novel enhanced vaccine strategy taking advantage of dendritic cells' ability to trigger antigen-specific immunity. Dendritic cells very efficiently process and exhibit on their cell surface antigen belonging to invading microbes, which then stimulates specific CD4+ and CD8+ cells to proliferate and attack the infection. The dendritic cells frequently move from the blood and mucous membranes to the lymph nodes, where T-cells concentrate.
The vaccine technique employs dendritic cells that have had part of the HIV gene set inserted into their nuclei. This quasi-infection enables them to produce HIV antigens internally. The HIV genes can be inserted using a recombinant, or hybrid, virus vector or by chemical means. The cells first are extracted from the body and cultured in the lab. When the genetic manipulation is finished, they are injected back under the skin.
One lab culture study 16 extracted dendritic cells from HIV-positive individuals and infected them with the same recombinant canarypox vaccine used in the Aaron Diamond trial involving patients treated during early infection. The induced T-helper cell and CTL responses marked an improvement over the results normally obtained with this vaccine.
In monkey experiments, dendritic cells modified by recombinant canarypox triggered notable CTL activity against HIV. 17 In one experiment 18 performed by the RIGHT Institute at Georgetown University, significant CTL responses to HIV lasted for at least seven months. The RIGHT Institute utilizes a chemical means of inserting the HIV genes. It now plans to work with an SIV construct that is pathogenic to monkeys and eventually to test the procedure on persons infected with HIV.
Even when stable and effective CTL defenses can be detected in patients, HIV can mutate and escape recognition by the CTLs. An analysis 19 of CTLs isolated from one patient by Bruce Walker's group at Massachusetts General Hospital found escape HIV mutants in both test-tube cultures and the patient's body, where they constituted 25% of the HIV population. These observations back up a recently published, much more extensive study of the way SIV, the simian version of HIV, mutates to escape immune defenses in three macaque monkeys. 20
This type of viral evolution is not commonly observed during HIV infection. There is no need for it because anti-HIV immune defense becomes strangely nonfunctional in many people. A French study 7 of the anti-HIV immune response detected a sizable number of HIV-specific CD4+ T-helper cells in six of 13 patients on long-term successful antiretroviral therapy. When cultured in the lab, the cells were able to begin secreting interferon-gamma when exposed to HIV proteins in the test tube, but they did not replicate. The cells from two of the patients could be pushed into action by adding the cytokine IL-12.
IL-12 is a cytokine released by macrophages. It promotes CTL responses by acting on the CD4+ T-helper cells that stimulate them. Among other things, IL-12 triggers increased IL-2 production by the CD4+ cells. Generation of IL-12 is inhibited by viral activity from the onset of HIV infection. 21 It is possible that successful immunization against HIV in people on HAART requires stimulating cytokines to supplement the vaccine.
One 40-person trial 22 tried this combined approach. British investigators added IL-2 and/or Remune after 16 weeks of successful HAART alone. They found that HAART alone did not lead to new immune defenses against HIV or a decline in HIV-infected cells beyond a certain point. HAART plus IL-2 at least accelerated that decline, and HAART plus Remune triggered the appearance of new anti-HIV CD4+ T-helper cells. In patients receiving all three, there was a tendency for this new anti-HIV immune response to appear after fewer immunizations with Remune.
1. Katlama C et al.ILSTIM (ANRS 082) – A Randomized Comparative Open-Label study Of Interleukin (IL2) In Patients With CD4 <200/mm3 despite Effective HAART. 7th Conference on Retroviruses and Opportunistic Infections (7th CROI). Jan 30-Feb 2 2000; poster 543.
2. Kamoto Y et al. Increasing Thymic Output with Exogenous IL-7. 7th CROI. Jan 30-Feb 2 2000; poster 326.
3. Fry TJ et al. A Potential Role for IL-7 in T-Cell Homeostasis in HIV Infected Patients. th CROI. Jan 30-Feb 2 2000; latebreaker 1.
4. Stellbrink HJ et al. Influence of Interleukin-2 (IL-2) on Productive and Latent HIV Infection and on Viral Rebound. 7th CROI. Jan 30-Feb 2 2000; oral presentation 240.
5. Hatano M et al. Plasma Viral Loads Approximate Pre-HAART Levels after Discontinuation of HAART. 7th CROI. Jan 30-Feb 2 2000; poster 349.
6. Ruiz L. Structured Treatment Interruption in Chronically HIV-1 Infected Patients after Long-Term Viral Suppression. 7th CROI. Jan 30-Feb 2 2000; poster 354.
7. Lafeuillade A et al. Aggressive HAART + IL-2 is Unable to Induce HIV Reemission in Early-Stage Disease after 18 Months. 7th CROI. Jan 30-Feb 2 2000; poster 544.
8. Prins JM et al. Immuno-Activation with Anti-CD3 and Recombinant Human IL-2 in HIV-1-Infected Patients on Potent Antiretroviral Therapy. AIDS. Dec 3 1999; 13(17); 2405-10.
9. Mittler JE and Perelson AS. Mathematical Model for the Decay of the Replication-Competent Latent Reservoir: Effect of Treatment Interruptions and IL-2 Stimulation. 7th CROI. Jan 30-Feb 2 2000; poster 349.
10. Charles A et al. Detection of HIV-1-Specific-CD4 T Cells by ELISPOT in Patients on Prolonged Highly Active Antiretroviral Therapy (HAART). 7th CROI. January 30-February 2 2000; oral presentation 579.
11. McNeil AC et al. HIV-Specific CD4+ T Cells are Maintained in Untreated Patients with or without Proliferative Response to HIV Antigens. 7th CROI. January 30-February 2 2000; oral presentation 186.
12. Gougeon ML et al. Immunological and Virological Efficacy of Long-Term IL-2 Therapy in HIV-Infected Patients. 7th CROI. Jan 30-Feb 2 2000; poster 383.
13. Lalazari J et al. Low-Dose Subcutaneous (SC) IL-2 in Combination with HAART Therapy Induces Significant Increases in NK Cells and Naive T-cells in Patients with <300 CD4+ T-cells/mm3: Results of a Randomized Controlled Trial MA-9801. 7th CROI. Jan 30-Feb 2 2000; poster 381.
14. Rizzardi G et al. Effect of HAART and Immune-Based Strategies in HIV-1-Infected Antiretroviral-Naive Adults. 7th CROI. Jan 30-Feb 2 2000; poster 545.
15. Jin X et al. Discontinuation of HAART After a Course of Therapeutic Vaccination with ALVAC1452 and rgp160 May Be Associated with Delayed Viral Rebound Kinetics. 7th CROI. Jan 30-Feb 2 2000; latebreaker 12.
16. Larsson M et al. Dendritic Cells Infected with Recombinant Canarypox Virus Induce Potent Anti-HIV Cytolitic and Helper T-Cell Response from Chronically Infected Individuals. 7th CROI. Jan 30-Feb 2 2000; poster 830.
17. Ignatius R et al. In Vivo T-cell Priming in Rhesus Macaques by Reinjected Immature and Mature Dendritic Cells Bearing Soluble or Recombinant Viral Vector-Encoded Antigens. 7th CROI. Jan 30-Feb 2 2000; oral presentation 434.
18. Lisiewicz J et al. Dendritic Cell-mediated Genetic Immunization Induces Vigorous T-helper and CTL Responses In Vitro and In Primates. 7th CROI. Jan 30-Feb 2 2000; poster 833.
19. Yang OO et al. Escape of HIV-1 from Cytotoxic T Lymphocytes in vitro. 7th CROI. Jan 30-Feb 2 2000; poster 602.
20. Evans DT et al. Virus-Specific Cytotoxic T-Lymphocyte Responses Select for Amino-Acid Variation in Simian Immunodeficiency Virus Env and Nef. Nature Medicine. November 1999; 5(11):1270-6.
21. Chambers KA et al. Suppression of Interleukin (IL)-12 Production by Acute HIV Infection and the Role of Other Cytokines. 7th CROI. Jan 30-Feb 2 2000; poster 572.
22. Hardy G et al. Effects of Combined Treatment with Interleukin-2 (IL-2) and an Inactivated gp120-depleted HIV-1 Immunogen (REMUNE) on Immune Reconstitution in HAART-Treated, HIV-1-Infected Individuals. . 7th CROI. Jan 30-Feb 2 2000; poster 341
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