MATHEMATICAL models and statistical evaluations are often used to explain biologic phenomena. The acquired immunodeficiency syndrome (AIDS) is no exception. Through the years of studying the human immunodeficiency virus (HIV) and its effect on the immune system, investigators have emphasized counting CD4 (+) cells and CD8+ cells and/or measuring p24 antigen levels or, more recently, the viral RNA content of the plasma. What is known, but not always appreciated, is the limited information that may be reflected by these parameters when measured in the blood. Peripheral blood lymphocyte numbers represent only a small percentage (3%) of the total white blood cells in the body, [1] and plasma virion content is just a portion of the total HIV virions, which are concentrated primarily in lymphoid organs. [2] Thus, in several experimental systems, plasma viral RNA may not reflect the actual viral load. Moreover, although CD4+ cell numbers may be helpful in predicting progression to AIDS, [3] changes in CD4+ cell levels do not appear to reflect consistently the clinical outcome of antiviral therapy. [4-6]
Because lower HIV RNA levels (ie, virus production) and higher CD4+ T-cell counts correlate with long-term survival in both adults and children, [7,8] clinicians prescribing antiretroviral drugs advocate these parameters as surrogate markers for successful therapy. However, a mistake can be made in assuming that drugs that induce such desired changes in these markers will produce the same clinical benefit. The physiologic reasons for these virologic and immunologic findings in an untreated infected individual can be quite distinct from those for such findings in a treated individual. In the former, a strong antiviral cellular immune response appears responsible. [9-11] In the latter, a block in de novo virus spread and replication is the most likely cause, not an immune system response. Studies have suggested that the extent of the anti-HIV immune activity can determine the equilibrium between HIV-1 expression and the asymptomat! ic period. [12,13] Thus, a beneficial clinical outcome cannot be expected unless drug therapies mimic the natural cellular immune responses that lower the viral RNA load and maintain CD4+ cell numbers. Most important, anti-HIV treatments may reduce the viral RNA load by blocking de novo infection, but unless they also decrease the established cellular virus load, the reservoir for renewed virus production remains and compromises the clinical outcome. [13]
In specific terms, counting peripheral blood CD4+ cells as a response to therapy can be misleading. Some clinicians, for example, would have us believe that an increase in CD4+ cells of 0.10 x109/L (100/microliter) with the administration of antiviral therapy indicates a successful approach. Recently, a gain in CD4+ cells of 0.009 x109/L per month over 6 months in subjects treated with zidovudine during acute infection was seen as an encouraging sign. [14] However, in a person with an uninfected needlestick injury we found a 30% increase in the number of CD4+ cells (0.25 x109/L) beginning within 2 days of antiretroviral drug treatment. [15] Such high levels were maintained for the full 4-week course of therapy. With cessation of treatment, the number of CD4+ cells returned to baseline. Redistribution of these cells from lymphoid sources, not necessarily! de novo synthesis of CD4+ cells, appears to be the mechanism for the increase in number. This nonantiviral effect of therapy needs to be considered in any analysis of efficacy. It may explain why changes in CD4+ cell levels during therapy have not correlated with survival. [4-6]
Likewise, to conclude that a 25%, 50%, or even 99% decrease in viral load levels in plasma indicates a promising approach to treating HIV infection is inappropriate. In virology, the effect of a treatment on virus replication should be measured in log or fold amounts; a percentage decrease or increase can be misleading. For example, 1 million RNA particles per milliliter of plasma, or about 1 billion virions in this total body fluid, has been reported as the content in blood of some symptomatic patients. [16,17] If antiretroviral drugs decrease the virus level 99%, this number indicates a 100-fold decrease. Thus, a reduction from 1 billion to 10 million circulating viruses is achieved in the plasma. Although this reduction seems dramatic, 10 million viruses can represent a significant number of viral variants that can replicate and spread in the host. Thus, the clinical benefit may be limited, since resistant strains can readily develop from this level of v! irus production. Another concern in viral load measurements is accuracy, since the variability of assays and persons can certainly make some changes (eg, 0.5 log) unreliable.
Any significant effort to control HIV must provide an extended period of greatly suppressed virus production. Most reports indicate, however, that viruses become resistant to current antiviral therapies within a few months after their initiation. [16-19] Recent evidence even suggests that resistance to the protease inhibitors is already present in viruses recovered from untreated individuals. [20] Moreover, with the widespread use of zidovudine, zidovudine-resistant strains are being observed during primary infection. [14] Thus, treatment directed at the virus alone is not sufficient. The key issue is the major feature of HIV pathogenesis: Unless the infected cell (the viral reservoir) is eliminated or its production of viruses is stopped, the virus will eventually prevail. [13]
A large reservoir of virus-infected cells (up to 250 billion cells) exists in the infected host. [2] Each cell can be a source of continual production of infectious particles or viral products toxic to the host. [13] Most studies indicate no effect of antiretroviral drugs on the level of these virus-infected cells in the blood or lymph nodes. [21,22] Certainly, a reduction in the number of these cells circulating in the blood (and in the lymph nodes) would be a more desirable indication of therapeutic potential than a decrease in the number of viral particles, most of which are noninfectious. [13] Serial biopsies of lymph nodes would be impractical, but studies, perhaps using flow cytometry, could measure reductions in virus-infected cells in the blood achieved by antiviral therapies. Ideally, these results would reflect as well the effects of therapy on infected cells in other parts of the body (eg, t! he brain and intestine).
In many ways, HIV infection resembles cancer, particularly the involvement of cells, be they infected or transformed. Therapy directed at the free virus alone, and not the cellular source of virus production, limits the chance of success. Approaches to control the virus-infected cell need to be developed, [13] particularly cell-mediated immune responses. Cellular viral load, drug toxicity, and the responsiveness of the host's immune system must all be considered, and the results of clinical trials need to be evaluated with an understanding of the number of individuals who did not complete the study because of drug toxicity or personal reasons. [23] This point becomes particularly important for another issue concerning numbers in AIDS research-survival time. In assessing the efficacy of antiretroviral drugs, can one really report a 50% increase in survival based on only 6 months of treatment and results that reflect 4.8% (treated) vs 8.4% ! (untreated) of the subjects studied? [24] A much longer period of observation and higher numbers of subjects treated need to be evaluated before drawing conclusions about such benefit of an antiretroviral therapy.
In therapeutic approaches, several aspects of HIV biology need to be appreciated. Inhibition of new virus infection by drugs for only a limited time may not be sufficient to bring a clinical benefit. Once a virus escapes the treatment, it can spread rapidly through the host and bring the pathogenic condition back to a state that resembles acute infection, perhaps even worse if toxic effects of therapy are involved. Developing effective anti-HIV drugs should remain an important part of our approach to treating AIDS but must be linked to efforts to enhance antiviral immune activity. In some cases, perhaps the anti-HIV drugs will have a direct beneficial effect on immune response. [25] Moreover, those therapies that improve quality of life should receive close attention. Many patients treated with antiviral drugs have experienced longer asymptomatic periods, even though their survival is not greatly increased. [26] Recent results with combina! tion therapies (eg, zidovudine and lamivudine) seem promising. [27] However, what is needed with antiviral drugs is at least a 1000-fold to 10 000-fold (not just a 100-fold) reduction in plasma viral RNA for years. Then, the chance of the emergence of drug-resistant viral strains or viruses that escape neutralizing antibodies would be greatly reduced. Moreover, such a result could help the host immune response against HIV.
Using antiretroviral therapies with immune-enhancing therapies could be the best approach for ensuring long-term survival of persons with HIV infection. If viral load were reduced effectively with antiviral drugs, ideally with no toxic effects, the immune system might better react to HIV to provide a long-standing, persistent antiviral response. Possible immune-based therapies that merit attention include the use of type 1 cytokines (such as interleukin-2 and interleukin-12), [28,29] inducers of cytokine production, [30] or activators of CD8+ cell antiviral responses. [30,31]
Thus, I envision antiretroviral drugs being used as adjuvants to immune-modulating therapies that would play the major role in AIDS defense by arming the host to combat HIV infection. Under these situations, virus production should be markedly reduced for long periods of time, and CD4+ cell function and number should be maintained. Long-term asymptomatic survival will be the ultimate result.
In virology and in other sciences in which biology plays an important role, statistical significance (even with a P value of .001) and mathematical formulas may not provide the desired insights into a problem unless the correct parameters are being measured. The many facets of biology and pathophysiology need to be considered. With HIV infection, the basic features of virus replication and pathogenesis, particularly the importance of the virus-infected cell, [13] must be appreciated. Medicine suffers when one is misled by numbers that are not relevant to the clinical problem involved.
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Acquired Immunodeficiency Syndrome; AIDS; Antiviral Agents; Biological Markers; CD4 Lymphocyte Count; CONTROVERSIES (Fontanarosa PB, ed); HIV Core Protein p24; HIV Infections; HIV p24 Antigen; RNA; Surrogate Markers; Viral Burden