Viral Dynamics of HIV: Implications for Drug Development and Therapeutic Strategies

The recent advances in our understanding of human immunodeficiency virus (HIV) burden, dynamics, and resistance provide an opportunity to reassess the theoretical and practical approaches to the development of HIV therapies. In the last decade, the design, conduct, and analysis of clinical trials of antiretroviral agents was hindered by an inability to directly measure HIV during therapeutic interventions. Changes in circulating HIV p24 antigen, detectable in only a few patients, were used to guide the development of new drugs. Mortality and the development of opportunistic infections were used to evaluate antiviral efficacy in larger phase III trials. The conclusions from these clinical studies were heavily influenced by an over-representation of patients at the highest risk for progression to these end points. As a result of these limitations, generalizations about the role of antiretroviral therapy in HIV disease evolved and paradigms for the evaluation of new drugs were established that are inconsistent with the current knowledge. The development of assays that detect and quantitate HIV in clinical specimens has permitted insights into viral burden, replication dynamics, and antiviral drug resistance in infected patients. These insights will expedite the development of new drugs and the design of therapeutic strategies and will support the implementation of earlier and more aggressive antiviral therapy for HIV infection. Viral Burden and Dynamics: Current Concepts Quantitation of viral infectivity in both plasma and peripheral blood mononuclear cells has confirmed that HIV infection is a remarkably dynamic process [1-8]. One week after the onset of clinical symptoms associated with primary infection, 100 to 10 000 infectious units of HIV can be detected per milliliter of plasma and per million peripheral blood mononuclear cells. Viral titers approach or exceed those seen in patients with advanced acquired immunodeficiency syndrome (AIDS). Within 1 to 2 months, the number of infectious HIV particles rapidly diminishes, often to levels below the limits of culture detection. This decline occurs in the absence of therapy, indicating either exhaustion of permissive host cells [9] or that the host immune response (probably cytotoxic T lymphocytes [10]) successfully suppresses much viral replication, at least temporarily. The rapid viral clearance and the resolution of the acute, clinical syndrome of primary HIV infection has supported the concept of a variable period of clinical latency after primary infection. This stage of infection is characterized by an absence of clinical symptoms, high levels of circulating HIV antibodies, and low or undetectable levels of plasma viremia and p24 antigenemia. Persistent replication of virus during this period can still be documented, however, through the isolation of virus from the peripheral blood mononuclear cells and cerebrospinal fluid of most patients at all stages of disease [11-13]. Recognition of the great magnitude and turnover of HIV replication during this period of clinical latency resulted from two approaches for detecting HIV nucleotide sequences in clinical specimens: in situ hybridization and quantitative assays for HIV RNA. Although electron micrographic and immunohistochemical demonstrations of ongoing HIV replication in the lymph nodes and central nervous system had been published during the preceding decade [14-22], in situ hybridization in lymphoid tissue graphically showed extremely high viral burdens at all stages of HIV disease, even in the absence of clinical signs and symptoms [23, 24]. If viral latency (defined as infection with nondefective viral DNA without productive virus replication) occurs in a subset of cells in the host, it is overwhelmed by persistent rounds of new infections of cells with high levels of replication even in the early stages of asymptomatic disease. During the asymptomatic stage, HIV RNA can be detected at high levels throughout the lymphoid system, especially in the perifollicular regions of the germinal centers [14, 17-2123, 24]. Lymph node hyperplasia develops, and immunohistochemical studies suggest that HIV-1, perhaps complexed by antibody, is trapped in lymphatic tissue by the processes of follicular dendritic cells [14, 19-2123, 24]. As many as one third of CD4 lymphocytes in lymphoid tissue and peripheral blood have detectable HIV DNA [24-26], much of which may represent defective genomes. A significant proportion of these CD4 lymphocytes, 0.1% to 1%, expresses HIV RNA at any given time, indicating active replication [24, 25]. Because such cells may have short lives, they represent a relatively small population but one with high turnover. In patients with higher CD4 cell counts, the proportion of infected lymphocytes in the lymphatic tissue is at least as great as and may be 5 to 10 times greater than that in peripheral blood mononuclear cells. This high proportion suggests that viral load replicates and increases predominately in the lymphatic tissue early during clinical latency [23, 24, 27]. Although synthesis of HIV RNA can be detected in no more than 1% of infected cells, the large reservoir of newly generated uninfected susceptible cells is sufficient to sustain the generation of substantial numbers of new cycles of replication and viral progeny. Several commercial assays are now available that quantitate HIV RNA in the blood using either genome amplification or signal amplification by branched-chain techniques [28-31]. These assays have different methods and sensitivity but produce similar results when applied to identical clinical specimens. The precision, reproducibility, and sensitivity of these assays have led to their use in place of or in addition to assays of viral infectivity in plasma and peripheral blood mononuclear cells for evaluating new antiretroviral treatments. Quantitative assays of viral infectivity are more costly, slower, less sensitive, and less precise than quantitation of HIV RNA; however, they generate observations concordant with HIV RNA assays and quantitate infectious virus, not just viral genomic material [32]. In HIV-infected cell culture supernatants and plasma, there are as many as 10 000 virions, each containing two copies of HIV RNA for every infectious particle [33, 34]. The quantitative RNA assay is thus more sensitive and has a greater dynamic range than assays of infectivity. With the use of assays that are based on genome amplification, such as polymerase chain reaction (PCR), almost every untreated patient infected with HIV has measurable plasma levels of HIV RNA throughout the course of disease (Figure 1) [35]. Approximately 105 to 107 HIV RNA copies/mL are present in the initial burst of viral replication that is associated with primary infection [33]. After 8 to 12 weeks, these levels are reduced approximately 100-fold with the development of the cytotoxic T-cell response [10, 33]. Published data are insufficient to correlate peak levels of plasma HIV RNA during primary HIV infection with subsequent clinical course, although symptoms during primary infection are associated with more rapid disease progression [36]. Figure 1. Relation between CD4 count and plasma level of human immunodeficiency virus (HIV) RNA level. Within 6 to 12 months after seroconversion, patients establish a steady-state level of plasma HIV RNA that varies from 102 to 106. These levels are highly predictive of subsequent disease course. Levels of HIV RNA tend to be higher in patients with more rapidly declining CD4 cell counts and symptomatic disease [33, 34, 37-39] (Figure 2). The viral burden in the few patients who escape CD4 cell decline and clinical progression for years (long-term nonprogressors) usually maintain fewer than 103 HIV RNA copies/mL [34, 40, 41]. Between these extremes are most patients, who maintain a range of 103 to 105 HIV RNA copies/mL and progress to AIDS in 8 to 10 years. Variability and exceptions exist, however, indicating that numerous factors (virus syncytium-inducing phenotype, other virus phenotypes, immune response, or immunogenetics) may contribute to varying natural histories. Figure 2. Schematic of human immunodeficiency virus (HIV) type 1 RNA levels and CD4+ lymphocytes during the course of infection. Top. Middle. Bottom. Plasma HIV RNA levels represent the steady state of a dynamic equilibrium between production and clearance of HIV RNA. Repeated measures over weeks or months show relatively constant plasma RNA levels in individual patients [33, 35]. The production of new virus is equal to the clearance of circulating virus. Immune stimulation through the administration of vaccine or new infections (such as influenza, reactivation of herpes simplex virus, or tuberculosis) produces transient increases in plasma viremia and HIV RNA levels [42-45]. The activation of CD4 lymphocytes by these processes probably generates additional permissive host cells for virus replication. Conversely, the administration of antiretroviral agents inhibits the production of new virus, thereby reducing the levels of HIV RNA. Reductions in viral burden in the lymph nodes may parallel reductions in plasma [46]. Shortly after the initiation of antiretroviral treatment, an exponential reduction of plasma HIV RNA permits the calculation of the viral clearance rates. In the studies of Ho and colleagues [47] and Wei and colleagues [48], plasma HIV RNA levels decreased exponentially during the first 1 or 2 weeks after initiation of therapy with several antiviral drugs [47, 48]. The estimated half-life of HIV in the plasma was 2 days. Thus, at least 30% of the plasma virus is replaced daily, implying that active de novo viral replication is primarily responsible for maintaining the level of plasma viremia. These rapid turnover rates are independent of the level of plasma viremia and CD4 cell count and are minimal estimates, because the calculations must assume complete inhibition of virus r