CUn. Immunother. 1996 Jul; 6 (1): 39-53

IMMUNOLOGICAL BASIS OF DISEASE

1172-7039/96/007-0039/$07.50/0 © Adis International Umited. All rights reserved.

Immunomodulatory Approaches to the Therapy of AIDS John L. Faheyl and Ronald Mitsuyasu 2 1 Departments of Microbiology and Immunology and of Medicine, Center for Interdisciplinary Research in Immunology and Disease (CIRID), Jonsson Comprehensive Cancer Center, UCLA School of Medicine, Los Angeles, USA 2 Department of Medicine, AIDS Clinical Research Center, UCLA School of Medicine, Los Angeles, USA

Contents Summary . . . . . . . . . . . . . . . . . . . . . . . .. .... ... . 1. Concept of Immunopathogenesis. . . . . . . . . . . . . . . . . . . 2. Laboratory Evaluations of the Effects of Immune-Based Therapies 2.1 Assessments of CD4+ T Cell Number and Functions 2.2 Measures of Immune Activation 2.3 Virus Quantitation .. . . . . . . .... . . . . . . . 2.4 Specific Immunity .. . . . . . . . . . . . . . . . . . . 3. Competence of the Immune System at the Time of Therapy 4. Overview of Immune-Based Therapies . . . . . . . . . . 5. Therapies Designed to Increase HIV-Specific Immunity . 5.1 Passive Immunity: Antibodies to HIV . . . . . . 5.2 Passive Immunity: Specific Cellular Transfer .. 5.3 Replacement or Augmentation of Stem Cells 5.4 Active Specific Immunisation: Vaccines . 6. Restoration of Defective Immune Function . 6.1 Interleukin-2 . 6.2 Interleukin- 12 6.3 Interleukin- 15 6.4 Interferon-a 6.5 InterferonI' . 6.6 Interleukin-4 . 7. Inhibitors of Inflammatory Cytokines . 8. Other Metabolic Approaches to Correct Malfunction 8.1 Antioxidants to Control Oxidative Stress . . . . 8.2 Reduction in Intracellular Cyclic AMP . . . . . 8.3 Growth Hormones for HIV-Associated Wasting 9. Gene-Based Therapies ... ... . . . . . . ... .

Summary

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Immune-based therapies are being explored on the presumption that host response to HIV is central to the pathogenesis and progression of HIV infection. Specific immune responses in most individuals serve to limit the acute stage of infection and induce a more chronic form of viral infection. Thus, immune re-

Fahey & Mitsuyasu

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sponse to HIV appears to be critical to virus control. On the other hand, mv infection induces widespread activation of immune cells, which in turn contributes to further HIV production. Also, immune dysfunction contributes to the development of a number of clinical disorders of AIDS, including opportunistic infections, wasting syndrome, AIDS dementia complex, Kaposi's sarcoma and lymphoproliferative disorders. Immune-based therapies in AIDS are directed at answering 2 major questions. Can specific HIV immunity be manipulated to better contain and reduce established HIV infection, and how can the pathogenic processes induced by HIV infection be reversed so as to maintain or restore normal function and health? The interventions explored thus far have been directed towards enhancement of specific immune mechanisms early in infection, restoration of defective functions and reduction of excessive activation secondary to cytokine derangement in HIV infection. Effective therapies developed to date include interferon-a for Kaposi's sarcoma and intermittent interleukin-2 (5-day courses every 8 weeks) to increase CD4+ counts. With greater understanding of the immunopathogenesis of HIV infection, better focused and potentially more effective therapies can be expected. Immune-based therapies are created from an understanding of the immunopathogenesis of HIV infection. As knowledge of pathogenesis increases, more effective means of controlling HIV replication and immune damage are developed. Immune therapies are also designed to maintain normal host immune functions and thus reduce the likelihood of opportunistic diseases . However, the immunopathogenic mechanisms underlying HIV infection and AIDS are complex and involve multifaceted interactions of the virus with the immune system.

1. Concept of Immunopathogenesis A brief review of the principal consequences of HIV infection will place in context consideration of therapeutic interventions. The natural history of HIV infection is outlined in terms of the major parameters of disease in figure 1. After initial HIV infection, the acute phase occurs where virus replication increases markedly with substantial viraemia. This is followed by cellular and humoral immune responses of the host which markedly reduce virus replication and viral load. However, the viral infection is not eliminated and viral replication becomes established in the lymphoid tissues. The continuing process of viral replication leads to decreases in CD4+ T cell numbers and immune impairment. Thus, viral load and viral cytopathic characteristics are major features of HIV pathogenesis.[1] Major elements of the immune pathology of HIV infection are outlined in table I. Specific im© Adis International Limited. All rights reserved.

munity that occurs in response to HIV infection is a second aspect of HIV pathogenesis. Both humoral and cellular immune responses occur against the initial HIV infection and dramatically reduce the viralloadP] However, the specific immune responses are unable to eradicate the virus, which remains active in lymphoid tissues of the body. Thus, the specific immune response has produced a shift from an acute to a chronic infection. Reduction in CD4+ T cell numbers is the third immunopathological process, one that was identified early as a unique feature of HIV infection! AIDS.[3] Apparently, HIV infection leads to shortened survival of newly formed T cells and the lymphoid system responds with increased CD4+ T cell production.[4,5] Compensatory increases in CD4+ production take place in most persons, and rapid turnover becomes the norm for CD4+ T cells. As long as CD4+ cell replacement keeps pace with removal, the CD4+ levels remain fairly stable (although at less than normal) or fall slowly. Immune Clln. Immunother. 1996 Jul; 6 (1)

Immunomodulatory Therapy in AIDS

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Fig. 1. Changes in median levels of the principal measurable disease parameters during HIV infection . In general, rapid course of illness is associated with greater abnormality in each parameter, e.g. lower CD4+ cell count and higher viral load and activation. Slower progression is associated with smaller changes. 'Activation' measures neopterin and ~2-microglobulin.

function (proliferation) is also decreased in HIV infection. [6) In most patients, the persistent viral infection sooner or later causes a decline in CD4+ lymphocytes. Later in the disease, virus replication increases either as a result of immune deterioration and/or as a result of loss of immunological control of HIV or as a result of changes in viral cytopathic capacity. In the end stages of the disease, many aspects of cellular and humoral immunity deteriorate with consequent occurrence of opportunistic diseases and, ultimately, death. Activation of immune cells and impaired function are the fourth immunopathological aspect of HIV infection. Activation occurs in CD4+, CD8+, B and natural killer (NK) cells and in monocytes/macrophages. This has been documented by: • increased serum IgG and IgA levels • increased plasma levels of several cytokines (and their receptors) and demonstration of increased cytokine mRNA expression in lymphoid cell populations • increased serum levels of neopterin and ~r microglobulin, which are products of activated macrophages and lymphoid cells • phenotypic changes on cell surfaces.[7-11) Furthermore, markers of activation (neopterin and ~2-microglobulin) have been shown to relate © Adis International Limited. All rights reserved.

to prognosis just as well as do the reduced CD4+ levels.[7) Cytokines are the messengers and effectors (regulators) of immune activation. HIV infection changes the production of cytokines throughout the immune system (table II). Plasma levels of cytokines reflect the stimulatory effects of HIV on the immune system. Separately, the capacity of the immune system to respond to new stimuli is impaired. This effect is evaluated by in vitro measures of cytokine production by peripheral blood mononuclear cells (PBMC) in response to stimuli such as mitogens or antigens. Thus, plasma levels of cytokines and induced cellular expression of cytokines reflect different aspects of the altered immunity that results from HIV infection. Dysregulation of the immune system is the functional consequence of the cytokine imbalances induced by HIV infection. A major contributor to this process is excess tumour necrosis factor-a (TNFa), which is generated primarily (but not exclusively) in monocytes. Interleukin (lL)-6 is another cytokine produced by macrophages which is increased in HIV infection)II) Excessive interferon-y (IFNy) production is a consistent lymphokine abnormality in HIV infection)IO) Because Table I. Pathology of HIV infection Specific immunity Inadequate response to HIV: cell-mediated immunity antibodies Immune deficiency Reduced CD4+ cell number and increased tumover Reduced interleukin-2 availability Increased cyclic AMP (suppressor system) Decreased glutathione (oxidative stress) Impaired immune function: dysregulation of responses to stimulation apoptosis of immune cells Activation Excessive cytokines: interleron-y tumour necrosis factor-a. interleukin-6

elln. Immunother. 1996 Jul; 6 (1)

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Table II. Endogenous cytokine changes in HIV infection Increased Tumour necrosis factor-a Interleukin-6 Interferon-I3-1 Interferon-y Decreased Interleukin-2 Not changed or unknown Interleukin-4 Interleukin-10 Interleukin-12 Interleukin-15

IFNy functions as a macrophage activation factor, it is likely to be an antecedent contributor to excessive production of TNFa and IL-6. In contrast, interleukin-2 (IL-2) gene expression is reduced, largely because of the reduction in CD4+ T cell number (CD4+ T cells are the principal source of IL-2). The overall reduction in IL-2 contrasts with the increase in IFNy)lO] Studies of lymphoid cell response in several non-HIV infections have revealed that the normal T helper 1 (THl) response, which occurs early in many infections (with IL-2 and IFNy cytokine response predominating), may switch to a TH2-like response. This is important in humoral immune responses, but can also elaborate a variety of cytokines, such as IL-4 and IL-IO, which may inhibit cell-mediated immunity. [1 2] Recently, in studies of the response of PBMC to various stimuli, Clerici and Shearer[!3] have suggested that HIV disease progression may be enhanced as a result of a THIto-TH2 switch. This hypothesis for HlV infection has not been fully substantiated and does not account for the high levels of pro inflammatory cytokines (such as TNFa) that are probably major contributors to immunopathology. Immune activation is central to the immunopathology of HlV infection, because activation is required for viral replication in infected CD4+ T cells.[!4] The mechanisms by which the cytokines increase HlV replication are varied (fig. 2). Cytokines may induce the production of a number of © Adis International Limited. All rights reserved.

Fahey & Mitsuyasu

proteins which act directly on the viral LTR (long terminal repeat region) to increase HIV replication. TNFa and IL-6, for example, have been shown to increase the elaboration of NFKB proteins, which in turn can bind to the HIV LTR promoter and increase HIV virus replication. Many of these inflammatory cytokines can also act at other sites, such as at the post-translational level, to increase the production of HIV proteins. Cytokine changes may also have direct bearing on clinical manifestations of HIV infection. Wasting syndrome, fevers, neurocognitive abnormalities (AIDS dementia complex) and the anaemia of chronic disease may also result from abnormal cytokine production in HIV-infected individuals. Work by Martinez-Maza and Miles at UCLA has indicated that Kaposi's sarcoma is a reactive tumour which proliferates in response to a number of HIV-induced cytokines, such as oncostatin M and IL-6)15] The lymphoproliferative disorders that are seen in association with HIV may also result from a number of B cell stimulatory cytokines produced as a result of HIV infection. Other metabolic changes have been noted in association with HlV infection. Reductions in cellular levels of glutathione[16] suggest poor cellular tolerance to oxidative stress. Increased levels of adenosine 3',5'-monophosphate (cyclic AMP) [intracellular suppressor systems] in lymphoid cells have been identified in patients with HIV infection)!?] Finally, increased apoptosis (programmed cell death) may result from metabolic disruption associated with inappropriate cellular activation and cellular dysregulation. [18]

2. Laboratory Evaluations of the Effects of Immune-Based Therapies Laboratory evaluation of the effects of immunebased therapies in AIDS is focused in 2 areas. The first is the effect of the therapy on the immune system. The effects of immune-based therapies will vary depending on the particular immune mechanism that is targeted for intervention. Therefore, appropriate markers for immunological efficacy Clin. Immunother. 1996 Jul; 6 (1)

Immunomodulatory Therapy in AIDS

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Activation and HtV infection

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Fig. 2. Schematic representation of the proposed sites of action of immune·based therapies for HIV disease. Cells, cytokines and virus involvement in immune activation and dysregulation and in viral production (left) and proposed loci of immune interventions (right). Abbreviations; CD4 CD4+ T cell; CDS CDS+ T cell; CMI cell-mediated immunity; IFNy interferon·y, IL interleukin; TNFa. = tumour necrosis factor-a..

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need to be carefully selected for each therapeutic agent. The second area includes assessment of drug effects on the pathogenic processes of HIV infection, e.g. CD4+ cell reduction, immune activation and viral load. Laboratory measures of these last parameters can be viewed, if suitably documented, as surrogate markers for the clinical efficacy of the therapy. 2.1 Assessments of CD4+ T Cell Number and Functions

Methods to measure CD4+ T cell numbers are improving. Flow cytometric methods that assess CD4+ number directly without separate white © Adis International Limited, All rights reseNed,

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blood cell and differential counts are adding to the precision of CD4+ testing. However, biological variables such as concomitant infections, diurnal variation and other factors can induce substantial variations in CD4+ numbers on a daily basis. Non-flow cytometric methods for measuring CD4+ T cells have been developed, and should prove adequate with well-trained personnel and good quality control procedures when the CD4+ counts are over 200/1l1 (200 x 1061L). These methods are likely to be introduced because of their lower expense and ease of use in the frequent testing done for assessment of immune-based therapies. Immune functions are progressively lost as disease advances. Loss of antigen-induced proliferaClin, Immunother, 1996 Jul: 6 (1)

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Fahey & Mitsuyasu

tive responses occurs initially to the least potent (microbial antigens), then moderate (alloantigens) and finally strongest (mitogens) stimuli.[l9] Functional testing is normally done by evaluating the proliferative response to mitogens and bacterial antigens. Proliferative responses to whole HIV or to viral components are variable and not generally useful for assessing immune status. Measurements of cytokine production in response to antigen stimulation also vary substantially, but can be used to document impaired immune function. 2.2 Measures of Immune Activation

The easiest and best documented markers of immune activation are the serum or plasma levels of soluble products of activation, such as serum neopterin or ~2-microglobulin.!7] Neopterin comes almost entirely from IFNy-stimulated macrophages and monocytes.f2°] ~2-Microglobulin reflects activation of a wider variety of cells and the effects of several cytokines.[21] Other markers of activation include soluble CD8 antigen and soluble cytokine receptors. Elevated serum levels of soluble IL-2 receptor (IL2Ra), however, do not have a strong relationship with prognosis.[22] Serum neopterin and ~2-micro­ globulin are lowered by antiretroviral therapy, and the relationship to clinical benefit is being determined. It should be noted that changes in these serum markers may reflect effects of therapy on lymphoid tissues throughout the body, not just in circulating cells. Phenotypic markers of activation on peripheral blood lymphocytes, such as increased CD38 expression on CD8+ T cells, have been shown to relate to prognosis in HIV infection.[9] Other studies have shown that CD38 levels correlate highly with serum neopterin and ~2-microglobulin levels.[23] Human leucocyte antigen (HLA)-DR, which is also increased in HIV infection, but which does not correlate well with CD38 expression, may provide additional information on immune response to HIY. The most direct measures of immune activation are the serum or plasma levels of cytokines (table © Adis International Limited. All rights reserved.

II). However, the levels are generally very low and in many instances, e.g. IL-2, IL-4, IL-lO, IL-12 and others, are below the level of detection in most individuals. Some cytokines, such as TNFa, IL-6 and IFNy, can be measured in HIV-seropositive individuals with some accuracy, especially in patients with more advanced disease. Cytokine gene expression can be measured by quantitation of specific cytokine mRNA in PBMC or in specific lymphoid subsets, such as CD4+ T cells. Sensitivity of cytokine measurements is enhanced through polymerase chain reaction (PCR) methods, where multiple amplification may increase the signal for the specific cytokine. Also, newer approaches to cytokine measurement, such as by branched DNA amplification techniques, are quite promising for measuring quantitative changes in cytokines during disease and with immune-based therapiesJ24] 2.3 Virus Quantitation

Plasma virus measurements are important in the diagnosis of HIV, and in assessing prognosis and evaluating the effects of treatment. In general, high viral load is associated with a poorer prognosis. Although wide ranges in viral load between individuals are found, reduction in viral load is a goal in all modes of therapy. Many methods have been used to detect and quantify HIY. Generally these methods can be divided into 3 groups: • serum p24 antigen quantitation • viral culture methods • nucleic acid hybridisation techniques. 2.3. 1 Serum p24 Antigen

HIV core protein, p24, in serum or plasma is considered to be evidence of HIV infection. The p24 antigen enzyme-linked immunosorbent assay (ELISA) is the simplest, fastest and least expensive HIV detection method. Most of the clinical research published before 1992 utilised methods that detected only free (unbound) p24 protein. The major limitation in using free p24 antigen measurement is that the majority of HIV-infected asymptomatic individuals have no detectable levels of free p24 antigen in the serum.[25] Even one-third of people with a diagnosis of AIDS do not have deClin. Immunother. 1996 Jul; 6 (1)

45

Imrnunomodulatory Therapy in AIDS

tectable free p24 antigen in serum. This limits its usefulness as a marker of changes in viral load due to treatment. Furthermore, when study patients are preselected for presence of p24 antigen, they are generally patients with more advanced or rapidly progressing disease. HIV p24 proteins can be present in the serum but totally bound to antibody, as was pointed out by Nishanian and colleaguesJ25 l The p24 antigenantibody immune complexes can be disassociated by acid pretreatment of the serum samples, which liberates the antigen, followed by measurement of immune complex-dissociated (lCD) p24 antigen. This assay increases the antigen-detectable positive population significantly, and is now commonly used to measure viralloads.[25l Also, the ICD p24 assay is useful in diagnosis of neonatal HIV infection.[ 26 l 2.3.2 Viral Culture

Viral quantitation by cell culture assay demonstrated that asymptomatic patients have significantly lower viral load than symptomatic ones. Coombs et alP?l reported that plasma viraemia is a measure for disease progression and is a more accurate predictor of progression than the presence of p24 antigen in plasma. 2.3.3 Nucleic Acid Hybridisation

PCR techniques have been introduced for the detection and quantitation of HIV nucleic acid. This powerful technique can amplify target DNA sequences (cDNA is a product of reverse transcribed RNA) that exist in extremely small quantities (e.g. as little as 1 copy per 106 cells). PCR methods enabled the development of rapid detection methods that are adaptable to clinical studies and are potentially able to be automatedp8l The branched DNA (bDNA) signal amplification method does not involve multiple cycles of replication as does the PCR technique, and may provide more exact HIV quantitationP9 l Measurements of HIV RNA in plasma[30l and in cells have demonstrated a correlation with the stage of HIV disease and with clinical progression. Also, reductions in viral load with antiretroviral drugs have been documented by this technique. © Adis International Limited. All rights reserved.

Induction of drug-resistant viral strains has been documented by serial analyses of viral isolates during clinical trials. Emergence of drugresistant HIV phenotypes and decline of therapeutic effectiveness is seen with all antiretroviral agents. A temporal correlation between the emergence of resistant viruses and an increase in viral load (free p24 and plasma viraemia) has been found for monotherapy with nucleoside reverse transcriptase inhibitors,l31l 2.4 Specific Immunity

Humoral immunity (antibodies to specific HIV antigens) can be readily assessed, but the relationship of antibody level to the course of disease is not very exact. Measurements have been made of antibodies to p24, gp41 and gp120, as well as to p 17 and reverse transcriptase and other antigens of HIY. Antibody levels may be higher in patients with more active disease[ 32l until the terminal stages, when antibody levels fall. A relationship to the benefits of therapy has not been established. Determination of antibody activity in terms of biological activity, e.g. antibody-dependent cellular cytotoxicity (ADCC) and anti virological effects, has not been particularly useful. Measurements of neutralising activity to virus infectivity, a measurement commonly used in virology laboratories, have not been found to relate to the course of illness if common laboratory strains of HIV were used,l33l The question is being re-examined in the context of autologous viral clones. Thus, antibody tests that show correlation with clinical benefit have yet to be established. Hopefully this will be possible because of the ease of measuring this aspect of immune response. Cell-mediated immunity, especially cytotoxic T lymphocyte (CTL) activity, is a cumbersome test at the present time. Growth of autologous B cells and insertion of an HIV viral component by means of a suitable vector are required. A general relationship has been established between CTL activity and stage of disease. However, because of the extensive cost of current CTL assays, this method is likely to be used only in selected clinical studies. Clin. Immunother. 1996 Jul; 6 (1)

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Fahey & Mitsuyasu

3. Competence of the Immune System at the Time of Therapy Under many circumstances, a response to immunebased therapies is dependent upon the competence of the immune system. Experience with a wide variety of immune-based therapies has confirmed the reasonable assumption that the degree of immune system damage will have a bearing on the effectiveness of the intervention. A specific example of this is the response of Kaposi's sarcoma to treatment with interferon-a (IFNa). It has been well documented that clinical benefit is more evident in individuals whose CD4+ levels are over 200/lll than in those with lower levelsJ34] HIV infection has been divided into 4 major stages: (i) CD4+ T cells greater than SOO/Ill, with least damage to the immune system; (ii) CD4+ T cells between 200 and SOO/Ill, an intermediate group who may be asymptomatic or have minor opportunistic infections; (iii) CD4+ T cells between 50 and 200/lll, with susceptibility to opportunistic infections; (iv) CD4+ T cells less than SO/Ill, as a premorbid state. CD4+ T cell counts are not a precise means of staging HIV infection, but the measurement is widely available and, therefore, is frequently used. More robust means of assessment that combine activation markers (serum ~2-microglobulin or neopterin) with markers of immune deficiency (CD4+ T cells) to establish an index have been proposed,[35] but have not yet been widely implemented. In summary, 2 categories of parameters (or markers) are needed to assess immune-based therapies: (i) those that measure the immunological effects of the particular intervention, and (ii) those that evaluate the effect on the HIV infection itself.

4. Overview of Immune-Based Therapies

sembled diagramatically in figure 2. The infection of CD4+ T cells and macrophages, the activation of T cells by HIV and its soluble products, the production of macrophage-stimUlating factors (e.g. IFNy) and the production, in turn, of TNFa which induces HIV production are all illustrated. The major forms of intervention in HIV disease are also indicated in figure 2 with their putative sites of action. The immune-based therapeutic approaches described in the following section are outlined in table III. In the discussion of individual therapies that follows, it should be kept in mind that many treatments have been evaluated only in patients with severely damaged immune systems. An absence of effect in this group does not necessarily preclude effectiveness in other situations where the immune system has more resilience. An effort is made in the following discussion to be clear about the clinical conditions in which particular interventions have been evaluated. Table III. Forms of immune-based therapies Increase of specific immunity Passive immunity: antibodies to HIV Specific cellular transfer Replacement of stem cells: bone marrow transplantation Active specific immunisation: vaccines Restoration of deficient immune functions Cytokine manipulation: interleukin-2 interleukin-4 interleukin-12 interleukin-15 interferon-a interferon-y Cysteine- and glutathione-related compounds Inhibitors of inflammatory cytokines Inhibitors of tumour necrosis factor-a and interleukin-6 Other approaches to correct malfunction Antioxidants to control oxidative stress Reduction in inhibition by intracellular cyclic AMP

Many of the essential changes induced by HIV infection that are noted in section 1 have been as© Adis International Limited. All rights reserved.

Growth hormones Gene-based therapies

Clin. Immunother. 1996 Jul: 6 (l)

Immunomodulatory Therapy in AIDS

5. Therapies Designed to Increase HIV-Specific Immunity

Specific immunity to HlV is a powerful response to initial HIV infection. Both antibodies and cellular immune responses are believed to contribute to containment of acute HIV infection. Therefore, efforts to enhance and extend HIV-specific immunity have been a major focus of immune-based therapies. 5.1 Passive Immunity: Antibodies to HIV

Antibodies have been shown to cause ADCC of viral particles, lysis of virally infected cells in the presence of complement, neutralisation of primary HIV isolates and protection of human cells in HUscm mice from HIV infection. Antibody can also reduce the immunosuppression caused by viral components such as gp120. The overall premise is that patients with progressing disease may fail to make sufficient amounts of protective antibodies. Therapeutic sources of antibodies include plasma and immunoglobulin (HIV Ig) from patients with stable HIV infection, murine monoclonal antibodies, hybrid antibodies and human IgG antibodies. Apart from plasma and HIV Ig, most of these antibodies are produced in cultured cell lines and are directed against gp120 or gp41 antigens. Initial clinical studies with plasma and HIV Ig have shown few adverse effects but little evidence of improvement, except for transitory reduction of plasma viral antigen levelsP6.371 More definitive trials of HIV Ig are under way in children, where 10 times greater plasma levels of antibody can be reached than in adults with HIV infection. Also, trials with HlV Ig to reduce maternofoetal transfer of HlV are under way. Trials with monoclonal human antibodies have been initiated in adults, although little or no benefit was seen in initial trials of monoclonal murine antibodies.[ 381 However, because of the potential variety of target antigens, because the antibodies differ in biological activity and because genetically engineered molecules will allow testing of novel © Adis International Limited. All rights reserved.

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hypotheses, the final answer on monoclonal antibody treatment of HIV infection lies in the future. 5.2 Passive Immunity: Specific Cellular Transfer

Cellular therapies have focused on CD8+ T cells or, more specifically, on HIV-directed CTLs. A secondary benefit of CD8+ T cells may derive from the observation that activated CD8+ T cells can suppress HIV replication in CD4+ cells in vitro.[ 391 Activated CD8+ T cells from noninfected persons can elicit the same inhibition, thus indicating that the effect is not due to specific immunity. A soluble CD8+ cell antiviral factor (,CAF') has been identified, but not yet fully characterised. In an effort to increase the effectiveness of CD8+ cytotoxic T cells, autologous CD8+ cells have been stimulated ex vivo with HIV antigens, expanded with IL-2 and then returned to the patientJ401There is some evidence for long term persistence of these cells after return, but their effectiveness in suppressing HIV remains to be determined. 5.3 Replacement or Augmentation of Stem Cells

In order to rebuild the immune system, several efforts have been made to restore deficient immunity by providing a new immune system by bone marrow transplantation or stem cell infusion.[41 1 However, unless the viral load is reduced, the transferred cells rapidly become infected and their functions are impaired in a manner similar to the original HIV infection. 5.4 Active Specific Immunisation: Vaccines

Several vaccines have undergone preliminary clinical testing in patients with HIY. It is postulated that postinfection vaccination with HIV antigens may boost effective immune responses and thereby alter the course of HIV infection. Most of the work that has been done so far has focused on subunit recombinant envelope protein vaccines, e.g. gp160 or gp120, using laboratory strains of HIV, such as IIIB, LAV or MN isolates.! 421 1t remains to be deClln. immunother. 1996 Jul; 6 (1)

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Fahey & Mitsuyasu

termined whether such vaccines are effective in inducing protective immunity, or whether vaccines will need to be made from primary isolates for each patient. The HIV vaccine developed by Salk is substantially different and represents a lysate of the whole virus. The original preparations lacked the envelope gpl20 protein, although recent versions have been supplemented by envelope protein. The Salk HIV vaccine has been shown to be without significant toxicity[43) and is being evaluated in individuals with relatively intact immune systems, e.g. those with CD4+ counts over 500/111. Finally, parts of the HIV virus can be inserted into a variety of cells as a means of inducing continuing exposure to selected viral antigens. Autologous fibroblasts, B cells and other cells, and the use of bacterial vectors are being explored. Also, preclinical trials with the DNA of HIV-specific antigens are being developed. Unfortunately, no vaccine evaluated so far has shown strong evidence of clinical or virological benefit in individuals with existing HIV infection. At the present time, many of these approaches are still at the early stages of exploration. 6. Restoration of Defective Immune Function 6.1 Interleukin-2

Interleukin-2 is a T cell-derived cytokine which induces activation, proliferation and differentiation of T and B lymphocytes. It can increase depressed NK cell activity in HIV infection and improve cell-mediated cytotoxicity. Thus, use ofIL-2 to restore deficient immune function has an attractive rationale. However, daily intravenous injections of IL-2 were not notably successfuI,£44) although IL-2 can be given subcutaneously at relatively low dosages with some transient immunological improvement.[45) In contrast, intermittent continuous IL-2 infusions (up to 18 MIU/day for 5 days, repeated at 8-week intervals)[46) have induced sustained increase in CD4+ T cell numbers. A >50% increase © Adis International Lirnited. All rights reserved.

in CD4+ cell counts was seen in 6 of 10 patients with initial CD4+ levels >200/111. Greatest responses were seen in those with higher CD4+ counts and minimal viral load. There were improvements in several other immune parameters as well. In patients with CD4+ counts <200/111 there was increased viral activation, little immunological improvement and substantial toxicity, including capillary leakage syndrome, severe flu-like symptoms, hepatic and renal dysfunction, thrombocytopenia and neutropenia. Because of the tendency for this therapy to increase viral load, the investigators suggested maximising antiretroviral treatment during IL-2 therapy.[46) Preliminary findings indicate that a sustained increase in CD4+ levels (e.g. > I OOO/Ill) may be maintained by repeated courses of intravenous IL2 at 6- to 12-month intervals. The mechanisms by which IL-2 effects these changes are uncertain. IL-2 may cause improvement in the disordered homeostatic mechanisms that regulate the activation and differentiation ofT lymphocytes during HIV infection. IL-2 may also prolong the survival of T cells by altering programmed cell death (apoptosis) induced by HIV envelope proteins or by cytokines. Exogenous IL-2 may also correct the imbalance between decreased IL-2 and increased IFNy production that characterises HIV infection.[IO) Determination of the mechanisms of action of these pharmacological dosages of IL-2 is important for generalising the approach and establishing a more exact condition for its use in treating HIV infection. 6.2 Interleukin-12

IL-12 is an inducer of IFNy in T cells and NK cells, and enhances cytolytic activity of these cells. Studies of IL-12, however, have been handicapped by the lack of a sensiti ve assay for detecting IL-12 in the plasma of people with HIV infection. There are two views as to the status of IL-12 production in HIV infection. One is that there is a deficiency in production of this cytokine, as suggested by the reduced capacity to produce IL-12 in response to stimulation of mononuclear cells in vitro. Furthermore, other in vitro studies demonstrated that ILClin. Immunother. 1996 Jul; 6 (1)

Immunomodulatory Therapy in AIDS

12 addition restored HIV-specific cell-mediated immunity.[47] Proponents of this view would administer additional IL-12 as a means of increasing immunological competence. The alternative view is that IL-12 production is increased during HIV infection and that this contributes to the immune dysregulation in HIV infection, including the well-documented increased production of endogenous IFNy. Other cytokines produced by monocytes, e.g. TNFa, IL-6 and IL-l, are increased in HIV infection. If IL-12 is not increased, it would be an exception as it, too, is produced by activated monocytes. Thus, the alternative view is that therapies should be directed towards reduction of IL-12 levels. Critical clinical evaluation of these opposing views has not yet been accomplished. 6.3 Interleukin-15

IL-15 is a cytokine that seems to be most effective in conjunction with other immunostimulatory cytokines such as IL-2.[48] IL-15 can increase the proliferative response of PBMC, but only when they have been activated. The IL-15 receptor shares the a and ~ chains of the IL-2 receptor, but also has a unique chain. IL-15 affects most types of T cells and NK cells to increase IFNy production, and can promote TNFa production by monocytes. IL-15 therapy studies have not yet been reported. 6.4 Interferon-a

IFNa was the first biologically active compound to be studied in AIDS. IFNa has multiple effects in inhibiting HIV infection in T cells as well as monocytes, acting late in the viral replication cycle, perhaps at the level at which viruses are assembled and released from infected cells. The principal use for IFNa has been in the treatment of Kaposi's sarcoma. Indeed, the Kaposi's sarcoma tumour response to IFNa is most pronounced in those patients who have some degree of intact cellular immunity.[34] © Adis International limited. All rights reserved.

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6.5 Interferon-y

Excessive production of IFNy is characteristic of HIV infection.l IO] Yet, stimulated PBMC from patients with HIV infection produce less IFNythan normal in response to microbial stimulation. Suggestions for replacement therapy with IFNyare frequently made, particularly in the context of local control of infections, as in the lung to deal with tuberculosis or other respiratory infections. IFNy may prove to be useful in controlling secondary infection. Studies to date do not indicate a value for IFNy in the containment of HIV infection.l49 ] 6.6 Interleukin-4

IL-4 is an inhibitor of IL-6 production. In Kaposi's sarcoma, the production of IL-6 and oncostatin M are increased and these cytokines act as autocrine growth factors for Kaposi's sarcoma cells. [15] Thus, a trial has been undertaken to determine if IL-4 treatment could control the growth of Kaposi's sarcoma (ACTG 224). Preliminary results of this ongoing study have shown a decline in p24 ICD antigen in more than half of the individuals treated so far (S. Miles, personal communication). No changes in CD4+ cell percentage or number have been observed. These preliminary studies are being extended to patients with CD4+ counts of 100 to 300/f..Ll to determine if a more intact immune system would respond better to IL-4. IL-4 therapy presents an interesting challenge to the hypothesis that a T H1-to-TH2 switch plays a major role in the course of HIV infection. [13] If this switch is important, then IL-4 therapy would be potentially disadvantageous. So far there has been no evidence of accelerated disease after IL-4 administration. It has been suggested, however, that the THl-to-T H2 switch is not a critical factor in HIV disease progression.l50] These hypotheses can be further evaluated in clinical trials such as this one with IL-4.

7. Inhibitors of Inflammatory Cytokines Pentoxifylline is a xanthine derivative that reduces TNFa gene expression in monocytes stimuClln. Immunat her. 1996 Jul; 6 (\)

Fahey & Mitsuyasu

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lated by lipopolysaccharides. Trials of pentoxifylline in patients with advanced HIV disease (e.g. in Kaposi's sarcoma and in patients with CD4+ counts less than about 200/f..Ll) have shown reduction in TNFa cytokine mRNA in PBMC, but not in serum levels of TNFa, ~2-microglobulin or neopterin. No increases in CD4+ cell levels were observed.£511 Thalidomide has selective inhibitory activity on the production of TNFa by macrophages stimulated by lipopolysaccharides. Thalidomide has been shown to be useful for the treatment of erythema nodosum forms of leprosy and severe recurrent aphthous stomatitis associated with HIV infection.l 521 Sedation and skin rash are common adverse effects. Controlled trials are under way to determine the benefit of thalidomide on this and other HIV conditions. Also, analogues of thalidomide are being developed with the goal of finding some with increased anti-TNF activities and decreased toxicity. It has been noted that the teratogenic effect of thalidomide is due to a metabolite that is not formed in mice. Thus, analogues that do not develop this metabolite in humans would perhaps be more generally useful. Other approaches based on inhibition of receptor binding of TNFa are also being developed.

8. Other Metabolic Approaches to Correct Malfunction 8.1 Antioxidants to Control Oxidative Stress

Is oxidative stress important in the pathogenesis of HIV disease? This question is posed because glutathione is decreased in the circulating cells of many people with HIV infection. [161 Glutathione is one of the major intracellular reducing agents. Thus, cells with reduced glutathione have less reserve to deal with damage-causing reactive oxygen intermediates (free radicals) which are generated during cellular metabolism. Low levels of free radicals are necessary for several important immune functions, including the inflammatory response and defence against bacterial infection. Normally a balance is maintained between free radicals and antioxidants. There are several major classes of © Adis International Limited. All rights reserved.

antioxidants: enzymes such as peroxidases, sulphydryl-containing pep tides such as glutathione, phenolic compounds such as vitamin E (tocopherol) and carotenoids such as ~-carotene. Low levels of antioxidants have been found in HIV infection. However, whether this situation contributes to the progression of HIV infection has not been resolved. Several efforts to replenish sulphydryl-containing cysteine and glutathione have been explored clinically in preliminary studies. Procysteine, given orally at dosages of 500, 1500 or 3000mg 3 times daily, showed inconclusive activity, with some decrease in ~2-microglobulin but no change in viral 10ad.l531 N-Acetylcysteine 600mg 3 times daily was not found to have any effect on viral load or immunological measures.l 541 These studies were generally done in people with CD4+ cell levels in the range of 200/f..L1. They did not show whether such treatment would be useful in the earlier stages of disease, e.g. CD4+ cell counts >500/f..L1. 8.2 Reduction in Intracellular Cyclic AMP

Lymphoid cells of patients with HIV infection have increased levels of cyclic AMP,[171 possibly as a response to chronic activation. Downregulation of these excessive inhibitory pathways can result in improved immune proliferative function. Efforts to induce similar changes in patients with HIV infection are in progress (B. Hofmann, personal communication). 8.3 Growth Hormones for HIV-Associated Wasting

A study of the recombinant growth hormone IGF-l (mecasermin) in patients with CD4+ cell counts of 100 to 400/f..Ll showed no change in numbers or percentages of CD4+ cells, in p24 antigen levels or in IL-2 production by PBMC in response to stimulation with gp160 peptides.l 551 There was also no change in response to recall antigens or phytohaemagglutinin in vitro. Some fluid retention was observed, which the investigators believed acClin. Imrnunother. 1996 Jul; 6 (1)

Immunomodulatory Therapy in AIDS

counted for most of the bodyweight gain. No antibody was found to the growth hormone. In another study,[56] patients with an average llkg body weight loss in the prior year received growth hormone or placebo. Those receiving growth hormone had increase in weight, increase in treadmill performance, no increase in HIV viral load and mild adverse effects. Preliminary studies indicated some improvement of immunological parameters. Bodyweight loss in some HIV-seropositive patients is associated with gastrointestinal disease. It has been noted that considerable variation in acute bodyweight loss can occur. The mechanisms for these changes remain to be determined. Studies relating to wasting represent problems in evaluation when bodyweight gain is the sole parameter of efficacy. For example, megestrol ('Megace' ) and tetrahydrocannabinol , which are sometimes used for this indication, can cause water retention rather than increase lean body mass. Thus, lean body mass would be the better parameter to follow, and is currently being incorporated in randomised controlled trials of therapies for HIV wasting. There is also some evidence that wasting responds transiently to antiretroviral therapy, e.g. during the time when the virus load is reduced and adverse cytokines are better controlled.

9. Gene-Based Therapies Three major approaches to gene-based therapies are currently being explored.[57] One involves transferring information directly to CD8+ cells to improve their cytotoxic capacity against HIV, or to CD4+ cells for improvement of T cell help to facilitate the cytotoxic activity of CD8+ cells. A second approach is directed towards interfering with viral production in cells containing HIV, or making lymphoid stem cells (and their progeny) resistant to HIV infection. Most of these studies are in stages of exploration in vitro. A third approach, using naked DNA, is being tried in an effort to improve immune response to HIV antigen. Full assessment of the benefits of each of these approaches will await the results of planned clinical trials. © Adis International Limited. All rights reserved.

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Additional information on experience with immunomodulatory therapies in HIV infection can be obtained from other reviews.£58-60]

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36. Jacobson JM, Colman N, Ostorw NA. Passive immunotherapy in the treatment of advanced human immunodeficiency virus infection. J Infect Dis 1993; 168: 298-305 37. Stiehm ER, Mofenson L, Zolla-Pasner S, et al. Summary of the workshop on passive immunotherapy in the prevention and treatment of HIV infection. Clin Immunol Immunopathol 1995; 75 (I): 1-10 38. Hinkula J, Bratt G, Gilljam G, et al. Immunological and virological interactions in patients receiving passive immunotherapy with HIV-I neutralizing monoclonal antibodies. J Acq Immune Defic Syndr 1994; 7: 940-51 39. Walker CM, Moody OJ , Stites DP, et al. CD81ymphocytes can control HIV infection in vitro by suppressing virus replication. Science 1986; 234: 1563-5 40. Ho M, Armstrong J, McMahon 0 , et al. A phase I study of adoptive transfer of autologous CD8+ T lymphocytes in patients with acquired immunodeficiency syndrome (AIDS)-related complex or AIDS. Blood 1993; 81: 2093-101 41. Lane HC, Zunich KM, Wilson W, et al. Syngeneic bone marrow transplantation and adoptive transfer of peripheral blood lymphocytes combined with zidovudine in human immunodeficiency virus (HiV) infection. Ann Intern Med 1990; 113: 512-9 42. Redfield RR, Birx DL, Ketter N, et al. A phase I evaluation of the safety and immunogenicity of vaccination with recombinant gpl60 in patients with early human immunodeficiency virus infection. N Engl J Med 1991; 324: 1677-84 43. Turner JL, Trauger RJ, Daigle AE, et al. HiV-1 immunogen induction of HiV-1 specific delayed-type hypersensitivity. AIDS 1994; 8: 1429-35 44. Lane HC, Siegel JP, Rook AH, et al. Use of interleukin-2 in patients with acquired immunodeficiency syndrome. J Bioi Response Mod 1984; 3: 512-6 45. Teppler H, Kaplan G, Smith K, et al. Efficacy of low doses of the polyethylene glycol derivative of interleukin-2 in modulating the immune response of patients with human immunodeficiency virus type I infection. J Infect Dis 1993; 167: 291-8 46. Kovacs JA, Baseler M, Dewar RJ, et al. Increases in CD4 T lymphocytes with intermittent courses of interleukin-2 in patients with human immunodeficiency virus infection. N Engl J Med 1995; 332: 567-75 47. Clerici M, Lucey DR, Berzofsky JA, et al. Interleukin-12: important cytokine in HIV-I immunopathogenesis. Science 1993; 262: 1721-4 48. Giri JG, Andeson OM, Kumaki S, et al. IL-15 , a novel T cell growth factor that shares activities and receptor components with IL-2. J Leukoc BioI 1995; 57: 763-6 49. Lane HC, Davey Jr RT, Sherwin SA, et al. A phase I trial of recombinant human interferon-yin patients with Kaposi's sarcoma and the acquired immunodeficiency syndrome (AIDS). J C1in Immunol1989; 9: 351-61 50. Romagnani S, del Prete G, Manetti R, et al. Role of TH IffH2 cytokines in HIV infection. Immunol Rev 1994; 140: 73-92 51. Dezube BJ, Pardee AB, Chapman B, et al. Pentoxifylline decreases tumor necrosis factor expression and serum triglycerides in people with AIDS. J Acq Immune Defic Syndr 1993; 6: 787-94 52. Paterson DL, Georghiou PR, Allworth AM, et al. Thalidomide as treatment of refractory aphthous ulceration related to human immunodeficiency virus infection. Clin Infect Dis 1995; 20: 250-4 53. Lederman M, Georges 0 , Borum P, et al. L-2-0xothiazolidinecarboxylate (procysteine) inhibits HIV-I expression but not

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interleukin-2 secretion [abstract). Program and abstracts: IXth International Conference on AIDS; 1993 Jun 6-11; Berlin, abstr. no. PO-A28-068I de Quay B, Malinverni R, Lauterburg B. Glutathione depletion in HIV-infected patients: role of cysteine deficiency and effect of oral N-acetylcysteine. AIDS 1992; 6 (8): 815-9 Nguyen BY, Clerici M, Yarchoan R, et al. Immunologic effects of human insulin-like growth factor and recombinant human growth hormone in HIV-infected patients. AIDS Res Hum Retrovir 1995; II Suppl. \: S 164 Schambelan M, LaMarca A, Mulligan K, etal. Growth hormone therapy for AIDS wasting [abstract). Proceedings of the 10th International Conference on AIDS; 1994 Aug 7-12; Yokohama, 2: 35 Rohrbaugh ML, McGowan JJ. Gene transfer for therapy and prophylaxis of HIV-I infection. Ann NY Acad Sci 1993 Jun 23; 685: 697-712

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58. Stein OS, Timpone JG, Gradon JO, et al. Immune-based therapeutics: scientific rationale and the promising approaches to the treatment of the human immunodeficiency virus-infected individual. Clin Infect Dis 1993; 17: 749,71 59. Fahey JL, Schooley R. Status of immune-based therapies in HIV infection and AIDS. Clin Exp Immunol 1992; 88: \-5 60. Lederman MM. Host-directed and immune-based therapies for human immunodeficiency virus infection. Ann Intern Med 1995; 122: 218-22

Correspondence and reprints: Dr John L. Fahey, Center for Interdisciplinary Research in Immunology and Disease, Departments of Microbiology and Immunology, UCLA School of Medicine, Center for the Health Sciences, 10833 Le Conte Avenue, Los Angeles, CA 90024-1747, USA.

Clin. Immunother. 1996 Jul; 6 (1)