Although viral load testing is the standard for measuring the effectiveness of antiretroviral therapy, it is at best an indirect measure of changes in the immune system. Several assays are now in development that measure cellular immune response. These tests could lead to better ways of evaluating the clinical status of the immune system and provide greater insight into the mechanisms of immune reconstitution. Recently Alan Huff, a Houston HIV/AIDS activist, interviewed Alan Landay, PhD, about new developments in this area. Landay is a Professor of Immunology/Microbiology at Rush Medical College in Chicago.
HUFF: Researchers are now looking at CD4 [T cell] response to HIV antigens using certain blood assays that can [measure] such responses. What is the reasoning behind this research?
LANDAY: What we realize is that with the advent of the newer, more potent antiviral regimens, we can have a sustained impact on [HIV]. We can have some impact on the immune system, but it doesn't normalize the immune system entirely. So, immune -- if we want to say immune -- reconstitution is partial, at best. There is a clear need to bring back the immune system. I think what we have learned in studying people such as long-term nonprogressors in this disease is that the immune system clearly is a very potent tool and is a potent approach that allows long-term suppression of the virus in those natural situations. We also know, from studying [multiply] exposed uninfected individuals (one of the most common examples being the Nairobi prostitute cohort of Frank Plummer) that these people do develop anti-HIV immunity. When those women take a break from their normal profession, they actually then get infected from the lack antigenic exposure.
HUFF: I just read something about that -- they just tested positive.
LANDAY: Correct. So the fact is that the low-level antigenic exposure of HIV, although not enough to infect them, allowed them to develop some sort of host protective immunity. When that went away, then they became infected -- and so it's raising a whole question of understanding the virus and the host immune system interaction.
HUFF: From your perspective, is the major immune response against HIV a cellular response?
LANDAY: When we talk about cellular immunity, we have to understand who the players are in this cellular immune response. The antigen presenting cells [include] monocytes/macrophages that circulate in your blood. There is also a cell that has that same [antigen presenting] function called a dendritic cell. It is a cell with tentacles and processes and can be found in your skin and other tissues as well. These are cells that sit in your tissues and are the sentinels to protect against infection from an invading organism. They first see the virus when it comes into the host, either through the mucosal female genital tract or the rectal mucosa, through sexual exposure. They then take the virus and carry it off to the lymph tissue -- the lymph nodes. There they interact with CD4 T cells. The CD4 T cells then may become infected and die. Without intervention, a person goes into acute disease. We know that person perhaps loses the cells that are reactive, perhaps due to direct infection. It [also] may be due to indirect killing, which is called activation-induced cell death. That is related to an elaboration of [events involving] cytokines and other molecules.
HUFF: Does that include apoptosis [programmed cell death]?
LANDAY: Absolutely. [There's] also the potential for those cells to become what we call anergic. They don't die but may be modified such that they don't work appropriately. The final outcome can be that they actually [become] HIV-specific CD4 cells that proliferate and respond. Then the role of the CD4 cell is to essentially produce cytokines, a major one being interleukin 2 (IL-2). There is a lot of effort now to use IL-2 as a therapy. Chiron [Pharmaceuticals] has 2 major studies, the SILCAAT and ESPRIT* trials are ongoing to identify the role [of IL-2]. Then there is interferon gamma, which is another cytokine made by the CD4 cells. These are CD4 cell factors that are necessary to turn on the CD8 cells (cytotoxic T lymphocytes). A CD8 cell can directly kill a target through the elaboration of different molecules, or it can indirectly suppress virus either through the chemokines or other factors that block virus. Jay Levy has described the CD8 cell antiviral factor (CAF). So there are known factors and still unknown factors that might be suppressive to virus.
HUFF: So what are the clinical implications of this information?
LANDAY: When we evaluate patients on highly active antiretroviral therapy [HAART], we want to find out how the CD4 cells function. This was originally introduced to us and brought to the forefront by Rosenberg and Walker who were [published] in Science a few years ago. They showed a good correlation between viral load and the ability of the CD4 cells to respond to HIV in the test tube. When you had good CD4 function, your viral load was low. When you had poor CD4 function, your viral load was high. That's the indirect measure -- it's not the CD4 cell that's directly impacting viral load, it's the CD8 cell. What we're saying is you need the CD4s to get the CD8s to work. Then we can measure those CD4 cells. I don't know if you want me to go into depth of how we do that.
HUFF: I do.
LANDAY: Okay. Well, we have several ways of doing it. The "old fashioned" way has been around since the 1970s and is what we call a lymphoproliferative assay (LPA). We do it in my lab routinely. We take cells from the patient, isolate the cells and take out the lymphocytes. Then we take those lymphocytes, put them into a culture dish and add 100,000-300,000 cells, depending on the assay conditions. We then add the HIV antigen to stimulate the cells.
HUFF: How is that done specifically?
LANDAY: We take a tube of blood, spin it down and are able to pull out the lymphocytes and monocytes. We then put them in our culture dish (96-well plate) and add the stimulus of antigen, which could be HIV, CMV [cytomegalovirus], candida, or any antigen of interest to us. We are now using 2 other pathogens that are important to us: hepatitis C [HCV] and hepatitis B [HBV]. We want to have a wide range of antigens to look at the immune response. We then let that cook in a 37oC incubator with CO2 [carbon dioxide] just like it would be in the human body. We add radioactivity to those cells for 6 hours. Next we harvest them, put them on a filter paper mat, place them in a "seal-a-meal" bag and count the radioactivity. From this we can calculate the counts per minute in the wells and determine how these cells have reacted functionally.
HUFF: And that would be, I guess, a number called the Stimulation Index (SI)?
LANDAY: What we get is the counts per minute in stimulated [cells] over counts per minute in unstimulated [cells] and that creates the formula for calculating SI.
HUFF: Will we want to see, hopefully, a greater SI?
HUFF: That might be an indicator that possibly these CD4s have the ability to respond to HIV?
LANDAY: That's what we're trying to do. We're also developing newer assays using flow cytometry, where we can -- instead of having to isolate cells -- take whole blood and stimulate the cells [with antigen] directly in the blood. Then, using the flow cytometer, we can identify the CD4 cells. In a matter of 6 hours, as opposed to 7 days, we quantify the function of those cells by their abilities to make cytokines. So this is a rapid procedure called an "intracellular cytokines assay" being developed through Becton Dickinson. Hopefully in the future it will replace what we are doing with the LPAs to give us a more quantitatively robust assay for measuring CD4 function and response.
HUFF: Do you feel that in the next 5 or 10 years these tools may be used like viral load in trying to measure [immune response]?
LANDAY: Absolutely. We need to improve the tools or surrogate markers that we are using now to measure responses to therapy. Most of our patients respond rapidly with viral loads going undetectable. However, we don't necessarily have the ability to then determine who's going to fail virologically. Also, we need to use these tools to understand how the immune system is coming back. Because I think for the long term we need to know if we will be able to restore the immune system and, if it's not coming back, we may want to add immune-based therapies to the treatment regimen earlier. We may even want to start a person upfront with an immune-based therapy, depending on what the patient looks like at baseline.
HUFF: Like IL-2 possibly?
LANDAY: Exactly. And we may want to start people with IL-2 plus HAART when they first walk in the [clinic] door if we think that's going to be of some benefit to their clinical outcome. We are very interested in understanding how to restore HIV-specific immunity. We think that is critical for long-term, successful clinical outcomes in patients. Even though we have experienced short-term success with the [current] therapies, we know in the long run that we are not going to be able to manage patients in the chronic disease [state] because we are not eradicating [HIV].
LANDAY: It is not going to be possible to have a patient on these therapies for 20, 30 or 40 years given the side effects -- the lipodystrophies and mitochondrial toxicities -- and the [adherence] issues that we all know exist. Some combination of antivirals with immune-based therapies is going to be critical. The other question is could we give a recently infected person a boost with an immune-based therapy, before even starting antiviral therapy? In that case, would you want to use a therapeutic immunization to boost HIV immunity? Could you boost without giving them HAART or delay the start of HAART so that you do not use up your options with the development of resistance and side effects? Finally, could you turn somebody who is a patient with early disease into somebody who is more like a long-term nonprogressor by boosting HIV-specific immunity?
HUFF: For the last 4 or 5 years everyone has thought you have to be below 50 [copies/mL] in your viral load -- or even below 20, but what happens when you do not have [enough of] that antigen present?
LANDAY: We have seen that in patients for whom we suppress [HIV] completely, we actually reduce the immune response to HIV and so the CD8 response to HIV actually wanes. [Sufficient HIV] antigen is necessary to maintain that response. We have reached [a] dichotomy. On the one hand we want to be able to get rid of the virus, but on the other hand we want to be able to maintain the immune response. I think what is going to have to happen in the future is a true balance where we suppress virus and maybe boost immunity by exogenous antigen. Several folks are trying this by structured therapy interruptions (STIs) with autologous [one's own] virus. Folks like Bruce Walker's group, Tony Fauci's group at NIH, David Ho's group, and others have been doing this predominantly in patients [who were treated during] acute disease.
HUFF: What role do you think drugs like Remune [HIV-1 immunogen] could play?
LANDAY: I think the therapeutic immunization strategies [like with Remune] will need to be played out. I have always looked at auto-immunization [immunization using the patient's own virus] being more potentially problematic because of the possibility for viral load rebound. You also get the decline of CD4s and it isn't clear whether you'll lose everything gained by [allowing an STI]. Are those CD4s going to be able to bounce back again after re-initiating therapy? I think another approach is to give the therapeutic immunization -- before an STI. So instead of using the patient's own virus to stimulate an immune response, you use exogenous immunogens.
HUFF: Like Remune?
LANDAY: Remune or canary pox or DNA vaccines. I think all of those have to be tried out. Perhaps, they all have different activities.
HUFF: So possibly in the future, maybe in the very near future, will this type of work be more important to the clinician in the field?
LANDAY: Oh, absolutely. This is what we are doing today in the Immunology Committee of the AIDS Clinical Trials Group (ACTG). We are looking at all the new therapeutic vaccines and are going to use the STI as a way of evaluating the efficacy of this [approach]. The proof will be if we can increase the durability of the viral suppression. The best way of testing that is to take people off their antivirals in a controlled clinical trial. Not one that is going to go out there and wreck immunity at this point. It's not ready, yet. We need to learn more because, as we all know, people are doing nonstructured therapy interruptions. They are going off of therapies for a variety of reasons, whether they are just tired of all the drugs, the side effects, body habitus changes and so on. I think we need to learn from that. It's important not just to work in the academic ivory tower, but also in the real field. [Community doctors need] to understand what is going on immunologically and virologically when their patients decide to have a nonstructured interruption.
HUFF: It sounds to me like [the doctors] really need to have this type of research behind that decision.
HUFF: So people really need to have a better understanding of whether their immune systems will even respond to quitting therapy.
LANDAY: Yes. We do not know that [yet]. Given what we do know, even in the best situation of patients who have been suppressed below [quantification levels of] the most sensitive assay for 3 to 4 years, when we look in their cells and other compartments there is still virus. We are not eradicating this [virus].
HUFF: The reservoirs are there.
LANDAY: So I think the future [of HIV treatment] has to be some approach that combines the ability to suppress the virus with enhancing the immune system. I think it only makes sense. What you have asked me today is if there are tools to help the physician decide, in an educated way, how to use immune therapy. We are not there yet, but we are working hard to develop those new tools so that we can move beyond where we are from an immunology point of view -- beyond CD4 [cell counts] to some of these more functional assays that can be used in clinical management. The problem with many of the assays that we use in clinical trials is they are just not at the point where I would feel comfortable in saying they [should be] routine clinical tests.
HUFF: Thank you very much.
*ESPRIT is an NIH-funded study using Chiron's investigational product.
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