Sharilyn K. Stanley

Immunopathogenic Mechanisms of HIV Infection

Dr. Anthony S. Fauci (National Institutes of Health [NIH], Bethesda, Maryland): The immunopathogenic mechanisms of infection with the human immunodeficiency virus (HIV) are multifaceted and multiphasic [1]. Figure 1 shows the typical prolonged course of HIV disease. The complex nature of HIV disease involves various overlapping features, including persistence of viral replication, aberrant and persistent immune activation, cytokine secretion and dysregulation, and, ultimately, progressive immunologic deterioration [1, 2]. Figure 1. Typical course of human immunodeficiency virus (HIV) infection. The New England Journal of Medicine Soon after HIV enters the body, it is widely disseminated, predominantly to lymphoid tissues [1-4]. The burst of virus replication early in the course of HIV disease is partially, but not completely, contained by an appropriate immune response [5-7], together with trapping of virions in the lymphoid tissue (see below). The onset of a robust immune response leads to a marked downregulation of virus in the blood [7, 8]. However, with rare exceptions, virus is not completely eliminated from the body, and a state of chronic, persistent viral replication ensues. In almost all other viral infections of humans, virus either kills the host within a short period (this is relatively rare), is completely eliminated from the body (this is the case with most viral infections), or enters a state of microbiological latency (this often occurs with herpes simplex virus infections). This transition by HIV from acute to chronic infection with persistent replication of virus (see below) is unique among viral infections in humans. We have previously shown that the lymphoid tissue is the major reservoir for and site of persistent viral replication; this is true even early in the course of infection, during the period of clinical latency when the CD4+ T cell count is only moderately decreased [3]. With the availability of sensitive assays for plasma viremia, it became clear that plasma viremia could also be measured at every stage of HIV infection, including the early asymptomatic stage [9]. Most recently, it has been shown [10, 11] that virus is present at high levels in the plasma and rapidly turns over, particularly in advanced-stage disease. In this setting of persistent viral replication, progressive deterioration of immune function usually occurs, ultimately resulting in profound immunosuppression and clinically apparent disease [3, 5, 6]. The link between the persistent replication of HIV and chronic activation of the immune system is critical to the pathogenic events seen in HIV disease [1, 12, 13]. In the early stage of HIV infection, the lymph nodes of persons with progressing HIV disease are activated and hyperplastic, and many virions are trapped in the germinal centers of lymph nodes in an extracellular manner on follicular dendritic cells [3, 4]. This occurs when production of virus by individual cells within lymphoid tissue is low [3, 14, 15]. Virus continues to be trapped by follicular dendritic cells in the germinal centers of the lymph nodes, initiating continuous immune stimulation [16] and constant exposure to possible infection of CD4+ T cells that reside in or are migrating through the lymph nodes [1, 2, 16]. In this regard, recent studies [17, 18] have shown that the HIV that is trapped on the follicular dendritic cells is infectious for CD4+ T cells, even though the virions are coated with neutralizing antibodies. Thus, the mechanisms operable in an appropriate immune response to HIV, particularly activation of the immune system, are paradoxically the same mechanisms that propagate HIV infection and lead to the ultimate destruction of lymphoid tissue and to profound immunosuppression [1]. Cytokine secretion is closely linked with the phenomenon of generalized cellular activation. Since the mid-1980s, our laboratory has studied the role of cytokines in the pathogenesis of HIV disease [19]. Cells communicate with each other through the secretion of cytokines as part of normal immunoregulatory homeostatic mechanisms [20]. During HIV infection, cytokines are hyperexpressed and, in some cases, dysregulated. Constitutive expression of cytokines was examined in the HIV-infected lymph node, where virus-infected cells reside, to determine their potential physiologic relevance. The constitutive and induced expression of various cytokines was assayed by polymerase chain reaction (PCR) [21]. At early, intermediate, or advanced stages of disease, it was found that interleukin-6, tumor necrosis factor-, interferon-, and interleukin-10 were over-expressed in the lymph nodes of HIV-infected persons compared with the lymph nodes of persons with other diseases. In contrast, interleukin-2 and interleukin-4 were rarely secreted constitutively at any stage of HIV disease, despite a state of persistent immune activation. Previous studies of chronically infected monocyte and T-cell lines [22-24] showed that cytokines such as interleukin-1 , interleukin-6, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor- can upregulate HIV expression. These observations assume potential physiologic relevance in light of the fact that several proinflammatory cytokines capable of inducing HIV expression are chronically overexpressed in the lymphoid tissue of HIV-infected persons [21]. We have recently shown that a tightly controlled autocrine loop of endogenous cytokine control of HIV expression exists [25]. In this regard, inhibitors of cytokine expression can markedly downregulate the expression of HIV in an in vitro infection model. Hence, the expression of HIV in vivo is probably at least partly modulated by the endogenous cytokine network that is generally responsible for maintaining the homeostasis of the immune system. Virologic and Immunologic Events Associated with Primary HIV Infection Dr. Giuseppe Pantaleo (NIH): A substantial proportion (50% to 70%) of persons with primary HIV infection have a clinical syndrome of variable severity [5, 6, 8]. The symptoms associated with this syndrome are nonspecific and may include fever, sore throat, skin rash, lymphadenopathy, splenomegaly, myalgia, arthritis, and, less often, meningitis. The lack of specificity and the variable severity of the clinical syndrome may explain, at least in part, why most HIV-infected persons generally do not report the symptoms of primary HIV infection to the physician. However, delineation of the immunologic and virologic events associated with primary infection is important for several reasons. First, infection is established and virus is systemically disseminated during primary HIV infection. Second, during this period, the initial encounter between HIV and the immune system of the host occurs, and an HIV-specific immune response is generated. Third, although both cellular and humoral immune responses are detected early in primary infection, these HIV-specific immune responses fail to eliminate HIV completely (see above). This suggests that the immune response may be inadequate or that certain mechanisms of viral escape from the immune response may be operative. Cell-mediated and humoral immune responses specific to HIV have been detected early in primary HIV infection [5-726, 27]. The contribution of these immune responses to the dramatic downregulation of viral replication during primary infection has been debated. It is likely that both cell-mediated and humoral immune responses are important in the initial downregulation of HIV replication. The cell-mediated immune response consists predominantly of HIV-specific cytotoxic T lymphocytes and is critical in the elimination of virus-expressing cells; thus, it results in decreased virus production [26, 28]. The humoral immune response, composed of antibodies against different HIV proteins, may substantially contribute to the downregulation of viremia through the formation of immune complexes composed of virus particles, immunoglobulin, and complement (C prime) that may be trapped in the reticulo-endothelial system [16, 28]. The appearance of trapped virus in the follicular dendritic cell network of germinal centers in lymph nodes coincides with an increase in the levels of C prime binding antibodies. In contrast, neutralizing antibodies are detected only several months after seroconversion. Therefore, the downregulation of viremia during the transition from the acute to the chronic phase of HIV infection may result from the combined action of both cellular and humoral immune responses. To better characterize the cell-mediated immune response during primary HIV infection, we analyzed the T-cell receptor repertoire in peripheral blood mononuclear cells [27]. Both CD4+ and CD8+ T cells can be further subdivided on the basis of families of cells that are identified by a particular variable region of the chain of the T-cell receptor (V ). The entire spectrum of V families, which number 24, are referred to as the V repertoire of T cells. The V repertoire has been analyzed on peripheral blood mononuclear cell samples collected at different time points after the onset of symptoms by combining a semiquantitative polymerase chain reaction (PCR) assay and cytofluorometry. The analysis, done in 20 persons with primary HIV infection, showed three predominant patterns of perturbations of the V repertoire: major expansion in a single V , moderate expansions in more than one V , and no expansions or minimal expansions in one or more V families. We then determined the cell subset involved in these expansions and the antigen specificity and function of the expanded V cell subsets. Patient 1, who is representative of the group that showed a major expansion in a single V family, has been an ideal patient in whom to address these issues. Results from analyses of Patient 1 are shown in Figure 2. Peripheral blood mononuclear cell samples were collected at different time points (days 16, 20

Effect of Immunization with a Common Recall Antigen on Viral Expression in Patients Infected with Human Immunodeficiency Virus Type 1

BACKGROUND Activation of the immune system is a normal response to antigenic stimulation, and such activation enhances the replication of human immunodeficiency virus type 1 (HIV-1). We studied the effect of immunization with a common recall antigen on viral expression in HIV-1-infected patients, on the ability to isolate virus, and on the susceptibility to HIV-1 infection of peripheral-blood mononuclear cells (PBMCs) from control subjects not infected with HIV-1. METHODS Thirteen HIV-1-infected patients and 10 uninfected adults were given a 0.5-ml booster dose of tetanus toxoid. Studies were performed to evaluate changes in the degree of plasma viremia, proviral burden, the ability to isolate HIV-1, and the susceptibility of PBMCs to acute infection in vitro. Two patients underwent sequential lymph-node biopsies for the assessment of viral burden in these tissues. RESULTS All 13 HIV-1-infected patients had transient increase in plasma viremia after immunization, and the proviral burden increased in 11. These changes did not correlate with the base-line CD4+ T-cell counts. The lymph-node tissue also had increases in the proviral burden and viral RNA after immunization. The virus was more easily isolated from PBMCs from nine of the patients after immunization than before immunization. Despite considerable variability in the results, PBMCs from 7 of the 10 normal subjects were more easily infected in vitro with HIV-1 after immunization than before immunization. CONCLUSIONS Activation of the immune system by an ongoing antigen-specific immune response to an exogenous stimulus transiently increases the expression of HIV-1 and may enhance the susceptibility of uninfected subjects to HIV-1.

Detection Assays for HIV Proteins

This updated unit describes four assays for detection of HIV proteins including: (1) a quantitative ELISA, (2) a quantitative immunoblotting assay, (3) a qualitative immunofluorescence assay, and (4) a functional assay to measure virus‐associated reverse transcriptase activity as an indicator of viral production.

UNIT 12.5 Detection Assays for HIV Proteins

This updated unit describes four assays for detection of HIV proteins including: (1) a quantitative ELISA, (2) a quantitative immunoblotting assay, (3) a qualitative immunofluorescence assay, and (4) a functional assay to measure virus‐associated reverse transcriptase activity as an indicator of viral production.