Dendritic Cells in Drag: Inauspicious Debut for HIV Specific CTLs; New Concerns about IL-12; Neutralizing Approaches for "Bad" Cytokines; Parke-Davis Targets Cellular Factors
The Clinical Immunology Society's 2nd HIV Immune-based Therapies Workshop was held in Baltimore on January 27-29. Attendance seemed a little down compared to last year's meeting, which may simply reflect the increased representation of issues pertaining to IBT development at other recent large AIDS meetings.
The meeting was commenced with keynote presentations by immunolgists/NIH AIDS scientific agenda-setters Bill Paul, director of the Office of AIDS Research (OAR), and Tony Fauci, director of NIAID. Following a customary description of the OAR, its mandates and its plans (coordinating NIH AIDS spending and "supporting investigator-initiated research", respectively), Dr. Paul segued into his scientific talk which centered around the phrase "optimal protective immunity," and the speaker's assertion that a preventive vaccine, in spite of current setbacks, represents the likeliest means of containing HIV infection in its global setting.
Of course, the phrases "optimal protective immunity" and "preventive vaccine" mandated the concomitant use of phrases like "new strategies" and "future directions", which is purely a function of markedly uncertain present directions, and abysmal old stategies. Paul stressed the importance of identifying and characterizing naturally attenuated strains of HIV, such as those believed to explain the long-term non-progressor states of individuals in an Australian cohort of blood transfusion recipients, and possibly the natural immunization of several groups of female sex workers in Africa .
Attention was also paid to recent experiments by Ron Derosier wherein rhesus macaques were protected from challenge with a pathogenic strain of SIV following inoculation with a nef-deleted attenuated version of SIV. Paul cited, however, new findings that an "attenuated" strain of SIV with three genetic changes (nef, vpr and NRE-deletions) caused simiam AIDS in 2 out of 3 newborn macaques. The results of this experiment underline our current, relatively unsophisticated understanding of HIV/SIV's genes and their functions-- not to mention how those genes might recombine once tampered with; and the fact that much more needs to be learned before we can even think about developing live-attenuated prophylactic vaccines.
Some interesting new data presented by two researchers from the NIH may bring us closer to the use of dendritic cells (DC) in the design of novel approaches to prophylactic and therapeutic HIV vaccines. DC represent a very small subset of immune cells that are potent antigen-presenting cells (APC) known to be top-of-the-line inducers of T-cell responses to HIV. When HIV, or a part of HIV (like the bits used in HIV vaccines) enters the body, it is recognized by T-cells only when it has been presented to them by APCs. This process of antigen presentation and recognition is the central event in our bodies' immune responses to infections, and a crucial part of HIV vaccine design involves getting T-cells to "see" HIV antigens.
Similarly, in HIV-infected individuals, the progressive failure of the immune system to fight HIV might result from HIV-mediated defects in antigen-presentation by monocytes and macrophages: two more common types of APCs. This NIH research team has found a way to isolate and remove pools of immune cells (90% monocytes, 10% DC) from the blood which can be induced, outside of the body, with the use of an agent called calcium ionophore, to function and act like DC. In vitro, these calcium-ionophore treated cells have been found to effectively present HIV antigens to both CD4+ and CD8+ T-cells. Theoretically, it is possible that if the same effects could be achieved in a person, the T-cell responses engendered by the DC-like cells could provide that person with protection against HIV infection. Those T-cell responses could also aid an already HIV+ person's immune system's fight against HIV and disease progression.
CD8+ T-cells and Cytokines
Dr. Fauci presented some interesting new information as part of his ever-evolving 'host factors' talk, the meeting's second keynote address. Fauci?s talk concerns host immunologic mechanisms that are thought to either contribute to or control viral replication and pathogenesis in people with HIV infection. For instance, the cytokines TNF, IL-1 and IL-6 and IFN-gamma, which are produced by immune cells during HIV infection, have long been known to increase HIV production in cultures of primary cells (cells from actual people). A paper by Fauci and colleagues, coming out this month in the New England Journal of Medicine, will include evidence that viral replication in the test tube can be virtually switched off when these host factors are neutralized with monoclonal antibodies. Fauci also showed evidence that the increases in HIV production caused by these cytokines may be controlled in the early stages of infection by other facets of the immune response--including what Fauci has termed a
"CD8+ cell suppressor phenomenon." Dr. Fauci reported that the HIV-inductive effects of the above-mentioned cytokines could be demonstrated only when CD8+ T-cells were removed from culture. This "suppressor phenomenon" is apparently separate from, but may occur in tandem with, the cytotoxic (cell killing) activity of CD8+ cells.
Fauci reported that the suppressor phenomenon was supported by the cytokine IL-2, which has also been found to increase HIV production by infected CD4+ T-cells. According to the data, the addition of IL-2 to cell cultures increases both the amount of HIV produced by CD4+ T-cells, and the ability of CD8+ cells to suppress virus production. The effects of IL-2, therefore, can be beneficial only when CD8+ cell suppressor activity can be induced by IL-2 to a greater degree than viral replication which, he added, may only be possible during early infection. This may help to explain why patients in earlier stages of HIV infection who received IL-2 infusions in NIH trials had significant CD4+ cell increases, while patients with more advanced disease suffered sustained increases in their viral loads, and did not experience any concrete immunologic improvements.
Interestingly, the cytokine IL-12 was found to stimulate CD4+ cell proliferation and viral replication in vitro, but not the CD8+ suppressor factor. Also, whereas IL-2, as well as IL-1, IL-6 and TNF were found to cause increased virus only when CD8+ cells were removed from culture, IL-12 was found to boost virus in unfractionated cell cultures. This is quite interesting, as the rationale for IL-12 therapy for HIV is that it enhances, in vitro, the ability of CD4+ T-cells to secrete IL-2. It is probable, therefore, that the effects of IL-12 upon IL-2 production by CD4+ cells may be limited to CD4+ cells, and that the IL-2 produced mediates no stimulatory effects upon CD8+ cells.
Gene Shearer of the NCI, perhaps the earliest proponent of IL-12 for HIV, appeared to have internalized these data and other concerns (such as potential toxicity) about IL-12's clinical application in his talk at the meeting. In this regard, he emphasized his findings that the inhibition of other cytokines, particularly IL-10, with monoclonal antibodies could effect many of the same immunologic changes as the addition of IL-12 in vitro, and that anti-cytokine therapy may prove to be a safer approach to immune modulation in humans. All of this withstanding, it should be noted that Fauci's new IL-12 data is thus far only relevant to a single in vitro system, and no researchers present took these findings as particularly gloomy news.
IBTs in Early vs. Late Disease
Brenda Lein of Project Inform's Project Immune Restoration (PIR) opened Saturday's scientific sessions with a tribute to the late Jessie Dobson (Founder of PIR), a description of her Project and one of its fundamental mandates: to stimulate and support research of immune reconstitutive therapies for late-stage HIV infection. Although a number of subsequent presenters paid lipservice to the importance of developing treatment modalities for persons with advanced disease, it was clear from the workshop's presentations (indeed, in all of IBT research) that little work is being conducted in that direction. Mike McCune, a prominent and innovative AIDS immunologist, summed up what is felt by many to be an unfortunate reality: That the majority of current approaches to immunotherapy rely upon a certain degree of baseline immunocompetence, and that most of the promise held by IBTs is in the treatment of HIV infection at earlier stages of disease.
This generalization more-or-less applies to IL-2 therapy, therapeutic vaccines and cell-transfer therapies, which require, it seems, some degree of immunocompetence and/or lower viral loads at baseline. Agents designed to inhibit inflammatory cytokines, IL-1, IL-6 and TNF, are, however, being studied in persons with late-stage HIV infection. In addition, IL-4, thought to be a "bad" cytokine in early infection, has been associated with significant decreases in viral burden in patients with below 100 CD4+ cells/mm3 at baseline who were treated with the cytokine as an experimental KS treatment. Steve Miles of UCLA is currently following up this surprising finding.
Novel Drug Targets Cellular Factor
A researcher from the CDC presented preclinical data on two related compounds donated by Parke-Davis Pharmaceuticals that inhibit activation-and TNF-induced viral expression by HIV-infected monocytic and T-cell lines. The usefulness of investigating molecular agents that target cellular factors used in the production/assembly of HIV has long been touted by AIDS researchers, including Dr. Fauci. While HIV is able to quickly alter its DNA (or survive in genetically altered forms) upon exposure to drugs that target its enzymes (like RT and protease) and proteins (eg, gp120), our bodies' cells do not. And because HIV relies upon many host cell proteins and mechanisms (some known, some unknown) to complete its life-cycle, cellular factors may prove to be easier, less mutable targets for anti-HIV drugs. These two agents, called PD121 and PD144 have been shown to dramatically suppress virus production by an as yet unknown mechanism of action which is able to return activated, virus producing cells to a relatively
quiesent state. What distinguishes these compounds from other novel compounds with in vitro anti-HIV activity is that Parke-Davis has already conducted pharmacokinetics and toxicology studies with these agents, which, in conjunction with antiretroviral assays, appear to warrant further investigation. It is likely that more information on the compounds' mechanisms of action will be required before more serious attention is paid to them.
As IL-13 is to IL-4 (that is, very similar), so is IL-15 to IL-2. This newly discovered cytokine, which is being preclinically evaluated by Immunex Corp., has been found to mediate many of the same immunologic functions as IL-2. IL-2 and IL-15 are both T-cell growth factors, and both potentiate antigen-specific T-cell proliferation in vitro. Both cytokines synergize with IL-12 to induce increased IFN-gamma expression, and both induce natural killer cell activity. IL-15 does not, however, stimulate resting T-cells in the way that IL-2 can in higher concentrations. For this reason, IL-15 may be more feasible than IL-2 as an adjuvant for vaccines, as global T-cell induction and the increased cytokine expression that goes with it would not be a risk. In other words, IL-15 has a much more selective effect than IL-2, and would only add fuel to antigen-driven T-cell responses. In this regard, IL-15 has been correlated with 6-fold fewer incidences of pulmonary edema, a possible consequence of massive lymphocyte expansion and
localization in the lungs, than IL-2 in mice.
Adoptive Cell Transfer
Several years back, Dr. Phil Greenberg and colleagues from the University of Washington in Seattle were able to prevent the common and life-threatening occurence of CMV infection and disease in immunosupressed bone-marrow transplant recipients by infusing them with billions of CMV-specific cytotoxic T-lymphocytes (CTL) generated from smaller numbers of such cells donated by a compatable (HLA-matched) donor. This success has led Dr. Greenberg, his colleagues and others to investigate the potential role for similar adoptive cell transfer therapies in HIV infection. Dr. Mark Gilbert presented results from the Seattle team's recent trial wherein HIV gag-specific CTL were isolated and genetically modified with a marker gene and a ?suicide geneÓ (which allows for the termination of infused cells if they start to cause problems). These cells were then multiplied into billions of cells and reinfused into their original donors. The results of the trial, which is not yet completed, were
less encouraging than those from the earlier CMV trial. No significant changes in CD4 or viral parameters were reported, and the cells appeared to have diminished survival after reinfusion, relative to the survival of cells in the CMV trial. The treatment appeared to be safe, with no major adverse vents occurring. The Seattle group plans to conduct follow-up experiments using similar techniques with the addition of concomitant IL-2 therapy to prolong the lives and improve the replicative capacity of the cells in vivo. Novel strategies, involving different types of genetic manipulation in the cells prior to expansion, are also being pursued by the group.
Monoclonal Antibodies as Antiviral Agents
Dennis Burton of Scripps Research Institute gave a thought-provoking and encouraging talk on what is essentially a new way to discover, screen for, modify and manufacture promising anti-HIV agents: human antibodies. In his group's approach, the "best of the best" antibodies can be identified by looking in the blood of long-term non-progressors (LTNP). It has been reported by Dr. Fauci and Dr. David Ho that the above-average antiviral activity of antibodies from LTNP contributes, at least in part, to the protection against disease progression observed in such individuals. Therefore, Dr. Burton and colleagues looked for (and found, in one LTNP) what they considered to be a most potent anti-HIV antibody. After locating the genes which encode for that particular antibody, they were able to get genetically engineered bacteria to make large amounts of it, which they called rIgGb12 (r=recombinant, IgGb=a class of antibody).
The antibodies, as expected, were good neutralizers of HIV in the test tube, but were found to be lacking against primary isolates of HIV (HIV from infected humans) in a mouse model of HIV infection. Not a problem. The researchers fiddled around with the genes for the HIV-binding region of the antibody until they identified a way to significantly improve its ability to neutralize primary isolates that the original antibody would not bind to. Not only did the "daughter" antibody neutralize more strains of HIV, it also neutralized the strains that its parent could bind with ten-times greater efficiency. This is truly a case of modern science improving nature's work.
The upshot of this new technique is that in the future, using combinations of such antibodies, it may be possible to provide every HIV-infected individual with an antibody attack against HIV better than that currently thought to be mounted by LTNP. Moreover, such antibodies may be an excellent tool in preventing mother-to-child infection. On the down-side, it is currently unknown how important the role of antibodies, no matter how good they are, is in controlling the infective process of HIV in the average HIV+ person.