Rationale for immune-based therapies for HIV-1 infection

REVIEW ARTICLES Rationale for immune-based therapies for HIV-1 infection HERNAN VALDEZ, LENA AL-HARTHI, ALAN LANDAY, and MICHAEL M. LEDERMAN CHICAGO,ILLINOIS Abbreviations: CTL = c y t o t o x i c l y m p h o c y t e ; G-CSF = g r a n u l o c y t e colony-stimulating factor; HAART = highly a c t i v e antiretroviral therapy; HIV-1 = h u m a n i m m u n o d e f i c i e n c y virus t y p e 1; IFN-v = interferon-y; IL-6 = interleukin-6; TNF-cx = tumor necrosis factor-e

eplication of HIV-1 occurs primarily in CD4+ lymphocytes and in mononuclear phagocytes, cells critical to host immune responses. In most infected persons, cell-mediated defenses become progressively and profoundly impaired. As is typical with intracellular pathogens, completion of the HIV-1 propagation cycle depends on efficient interaction with and utilization of numerous host-cellular elements. Moreover, it appears that immune activation enhances HIV-1 propagation. Thus the relationship between HIV-1 and immune function is intimate, complex, and, by most standards, difficult to unravel.

R

RATIONALE FOR IMMUNE-BASED THERAPIES

Although immune deficiency is the hallmark of infection with HIV-1, immune defenses--particularly cytotoxic T lymphocyte responses--are essential regulators of viral propagation after acquisition of infection,a and by inference are critical predictors of long-term prognosis. 2-4 Nonetheless, progressive depletion of cell-mediated immune responses, as characterized by losses in circulating and lymph From the Center for AIDS Research and the Divisionof Infectious Diseases, Case WesternReserveUniversity;and University Hospitals of Cleveland and the Rush Presbyterian St. Luke's Medical Center. Submittedfor publicationJune 10, 1997;revisionsubmiltedSept. 11, 1997; accepted Sept. 11, 1997. Reprint requests: MichaelM. Lederman,MD, Divisionof Infectious Diseases, UniversityHospitals of Cleveland, 11100Euclid Ave., Cleveland,OH 44106-5083. J Lab Clin Med 1998;131:197-206 Copyright© 1998by Mosby,Inc. 0022-2143/98 $5.00 + 0 5/1/87249

node T-lymphocyte populations and functional impairments of the remaining cells, are expected longterm consequences of HIV-1 infection. This immune dysfunction predisposes to the opportunistic infections and neoplasms that define AIDS. Despite profound immune deficiency, HIV-1 infection is also associated with evidence of immune activation: increased plasma levels of the proinflammatory cytokines TNF-o~ and IL-6, increased immunoglobulin synthesis and increased activation antigen expression on CD8+ as well as CD4+ lymphocytes.5'6 It is posited that dysregulated immune activation contributes directly to the morbidity of HIV-1 infection (wasting and autoimmune phenomena), and accelerates HIV-1 propagation. The recent introduction of HAART regimens has dramatically changed the outlook (at least in the short term) for HIV-l-infected persons] A decreased incidence of opportunistic infections and decreased short-term mortality are the consequences of these newer treatments. Presumably, these benefits are related to a cessation of virusinduced immune deterioration, or even to some degree of immunologic reconstitution. Preliminary data indicate that in the short term, immune reconstitution is modest after HAART, with an apparent ceiling to the increase in circulating CD4+ lymphocyte numbers, and that lymphocyte proliferation is enhanced primarily in response to antigens to which some degree of response has been retained. In addition, the profound perturbations in the distribution of lymphocyte T-cell-receptor V[3 families (a reflection of the potential diversity of antigen recognition by T lymphocytes) seen in some HIV-1 infected persons are not corrected or only minimally 197

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T a b l e I. Results o f s e l e c t e d p u b l i s h e d clinical trials of i m m u n e - b a s e d t h e r a p i e s for t h e t r e a t m e n t of

HIV-1 disease Ref

Design

Intervention

Laboratory outcome

Clinical outcome

No change in CD4+ cell counts. 4, in plasma HIVRNA levels of 0.02 and 0.65 logs in the 0.5 and 1 gm/day groups

ND

Nausea,diarrhea, stomatitis, neutropenia.

I' in LAK activity, NK activity, LPA to PHA. No change in CD4+ cell count, p24 antigen, or culturable HIV 16 patients with 1` in DTH responses, NK cells, CD4+ counts beCD4+ cells at high dose. No tween 200 and 500 change in plasma HIV-RNA

ND

Flu-like symptoms, myalgia, fever.

ND

Fever, myaigia, fatigue, worsening of asthma

Polyclonal I' in CD4+ cells. $ in cellular activation markers. Transient 1` in plasma HIVRNA

ND

Fever, flulike symptoms, capillary leak, rash

1' of 37 CD4+ Cells/month in IL-2 group. No difference in plasma HIV-RNA

ND

Fever, malaise, fatigue, 1' bilirubin

Transient J. in lymphocyte count, ADCC, NK cell number

ND

NR

TNF levels I' in Tenidaptreated patients

ND

Rash

Population

Interventions interfering with HIV-1 replication 18 Open label Hydroxyurea (0.5 or 1 26 patients with gm/day) + ddl for 4 CD4+ cells beweeks in patients tween 100 and with at least 3 350. months of ddl Treatment with T-helper cytokines 31 Open label 36,000 IU PEG IL-2 16 patients with ID twice weekly for CD4+ cell counts 4 months > 100

33 Open label, N A R Tand 62,500 to dose-finding 500,000 U/m 2 of lL-2 daily for 6 months 34 Open label, NART+6-18 MU/day 23 patients with dose-escaof IL-2 by OIV in CD4+ cell counts lation 5-day > 200 cycles every 8 weeks 35 Open label, N A R Tvs. NART+ 60 patients with controlled IL-2 by ClV starting CD4÷ cells > 200 at 18 MU/day in 5-day cycles every 8 weeks 45 Single dose, 30 to 1000 ng/kg of 15 patients with dose-escaIL-12 CD4+ cells belation trial tween 100-500 Inhibition of proinflammatory cytokine expression 56 Double blind, Placebo or tenidap 43 patients with placebo 120 mg/day for 6 CD4+ cell count controlled, weeks < 400 crossover 58 Double blind, Thalidomide 300 mg/ 39 patients with wastrandomized day or placebo for ing or TB 21 days 60 Double blind, Placebo or thalidorandomized, mide at 200 mg/ placeboday for 4 weeks controlled

62 Open label trial Pentoxifyiiine400 mg TID for 8 weeks 63 Double blind, placebo controlled

Placebo or pentoxifylline 600 mg TID for 4 months

66 Open label

10 to 40 mg/d of murine monoclonal anti IL-6 antibody for 2 to 3 21 -day cycles 10 to 40 mg/d of murine monoclonal anti IL-6 antibody for 21 -day cycles

67 Open label

Thalidomide-treated: > 10% $ Weight gain in in plasma TNF; $ in plasma thalidomideHIV-RNA in TB patients treated patients 60 patients with oral No change in CD4+ cells. In In thalidomide aphtous ulcers thalidomide group: 1' in treated: plasma HIV-RNA, TNF and 90% ulcer TNF receptors resolution and weight gain 25 patients with AIDS TNF mRNA and triglycerides ND on stable NART $ ; no change in HIV isolation from PBMC 120 patients with pul- In pentoxifylline treated: $ in Hemoglobin monary TB plasma HIV-RNA, TNF proI' and 1' duction, and beta2 microperforglobulin mance 11 patients with, $ In serum levels of C-reactive Lymphoma CD4+ cells < 100 protein progressed and lymphoma in 5 and stabilized in 6 patients 11 patients with Albumin 1', normalization of ND CD4+ cells < 100 C-reactive protein; no and lymphoma change in CD4+ or CD8+ cells or plasma HIV-RNA

Adverse effects

Rash

Somnolence, rash

Nausea, fever

Nausea

Platelet and neutrophil count $

NR

J L a b Clin M e d V o l u m e 131, N u m b e r 3

V a l d e z e t al,

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Table I. c o n f ' d Ref

Design

Intervention

Replacement of immunologically active cells 79 Phase I Infusion of ex vivoactivated and expanded autologous C D 8 + cells (108 to 101° ) 80 Phase I 5 infusion cycles of ex vivo-activated and expanded C D 8 + cells (108 to 10 ~°) followed by low dose IL-2 86 Phase I Autologous HIV-specific cytotoxic C D 8 + cells (108 to 109) modified to express HSV-TK and expanded ex vivo

Population

Laboratory outcome

7 patients with severe No change on C D 4 + or C D 8 + ARC or AIDS and cells, p24 antigen or HIV isopositive p24 antilation. Moderate I' in CTL gen or culturable activity postinfusion HIV 6 patients with severe No change in C D 4 + or CD8+ ARC or AIDS cells, or p24 antigen

6 patients with 200 to 500 CD4+ cells

Development of cytotoxic cell response against transduced, reinfused C D 8 + autologous cells in 5 of 6 patients

Clinical outcome

ND

Adverse effects

NR

Improvement in KS, OHL, adenopathy

None

ND

NR

Abbreviations: $ : decrease; 1' : increase; PEG = polyethylene glycol; LAK = lymphocyte activated killer cell; LPA = lymphoproliferative assay; PHA =

phytohemagglutinin; NK = natural killer; ND = not determined; NR = not reported; NART: nucleoside reverse transcriptase inhibitor; ClV = continuous intravenous infusion; MU = million units; ADCC = antibody-dependent cell-mediated cytotoxicity; PBMC = peripheral blood mononuclear cells; CTL = cytotoxic T lymphocytes; KS = Kaposi's sarcoma; OHL = oral hairy leukeplakia; HSV-TK = Herpes simplex virus thymidine kinase.

corrected by HAART, at least in the short term. 8'9 Perhaps more prolonged suppression of viral replication will permit increasing degrees of immune restoration, but the durability of antiviral therapies is limited by the emergence of viral escape mutants, and the likelihood of escape is promoted by the unforgiving clinical requirements for strict compliance with suppressive treatment regimens to ensure that viral replication is continuously blocked. In summary, HIV-1 parasitizes immune-competent cells, utilizes host-cellular elements and immune activation for its own propagation, and induces progressive, life-threatening immune deficiency that is only partly improved as a result of antiviral therapies. Given these conditions, the rationale for treatment trials of host-directed and immune-based therapies for HIV-1 infection can be summarized as follows: (1) Host-directed therapies may interfere with HIV-1 replication and may be less susceptible to the emergence of viral escape mutation. (2) Treatment with T-helper cytokines may enhance cell-mediated immune responses in persons with HIV-related immune deficiency. (3) Inhibition of proinflammatory cytokine expression may decrease the immune activation state that characterizes HIV-1 infection, and also may indirectly inhibit HIV-1 expression. (4) Replacement of immunologically active cells or their precursors may help restore depressed immune defenses in persons

with AIDS. (5) Well-designed trials of host-directed or immune-based therapies can not only permit examination of the utility of a treatment intervention, but can also provide an opportunity to test critical hypotheses of HIV disease pathogenesis. Host-directed therapies. Host-directed therapies may interfere with HIV-1 replication, and are less susceptible to the emergence of viral escape mutation. A rate-limiting factor in successful antiviral treatment strategies is the predictable emergence of viral escape mutations as a consequence of continuous, high-level viral replication and an error-prone reverse transcriptase. Because host elements are utilized for every phase of viral replication, interference with host-virus interactions may block viral replication. Mutational escape from these interventions is less likely, since the target elements are encoded on host genes. On the other hand, immunologic toxicities of host-directed therapies are predictable, but the redundancy of host-immune defenses may permit targeting of these elements without dose-limiting toxicity. Several potential targets for host-directed antMral treatment strategies include host nuclear factors that enhance HIV-1 transcription1°-12; cellular cofactors for Tat, the transcriptional regulatory protein that mediates high-level expression of HIV genes13-15; cellular chaperones for assembly of viral proteins16; enzymes responsible for the generation

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of dinucleotide triphosphates needed to assemble proviral DNAs17; and the recently identified chemokine receptors that serve as co-receptors for cell entry of both macrophage-tropic HIV-1 and lymphocytotropic HIV-1 isolates, lsa9 Recent trials of hydroxyurea, an inhibitor of ribonucleotide reductase, have enhanced the antiviral activities of dideoxynucleotides with tolerable host toxicities. 2° More recently, persons homozygous for a 32-bp deletion in the chemokine receptor gene that results in a failure of surface CCR-5 expression have been found to have an apparent relative resistance to acquisition of HIV-1 infection in vivo.21 This resistance is mirrored in cellular resistance to HIV-I isolates that are macrophage-tropic in vitro. 22 Most importantly, these persons, although relatively resistant to HIV-1 in vivo, are otherwise healthy and not immune-impaired. Thus, targeting of the CCR-5 receptor may block viral replication without deleterious effects on the host. Alhough the foregoing approach may prove useful, additional co-receptors may be used for HIV entry, 23 and introduction of a selection pressure against macrophage-tropic, CCR-5-dependent HIV isolates may facilitate emergence of CXCR-4-dependent, syncitium-inducing (SI) strains that are associated with a more accelerated disease course. 24 These considerations underscore the importance of carefully designed preclinical and clinical studies to assure that treatment strategies targeting host elements are developed in a rational and safe manner. Treatment with T-helper cytokines. Treatment with T-helper cytokines may enhance cell-mediated immune responses in persons with HIV-related immune deficiency. Cytokine dysregulation may play an important role in HIV-1 disease pathogenesis. Cytokine profiles are altered early in HIV disease 25 and expression of T-helper cytokines is decreased during the course of HIV-1 disease, 262s and this decrease may contribute to the functional impairment in immune responses seen in HIV-1 disease. Administration of T-helper cytokines may help correct these impairments. Interleukin-2. IL-2 broadly enhances immune responsiveness; it induces T-cell proliferation, enhances cell-mediated cytotoxicity, and also enhances antigen presentation through increasing cell-surface human leukocyte-associated antigen DR (HLADR) expression. 29 In vitro, IL-2 partly corrects the impaired lymphocyte NK cell activity and impaired CD8+-lymphocyte cytotoxicity in patients with A I D S , 3° and the increased tendency of lymphocytes to undergo apoptosis 31 seen in HIV-1 disease. In addition, IL-2 enhances CD8+-lymphocyte-medi-

J Lab Clin Med March 1998

ated suppression of HIV-1 replication. 32 Because IL-2 production and IL-2 receptor expression on CD4+ lymphocytes are decreased in HIV-1 disease, 26'27 trials of administration of this cytokine to HIV-infected patients are warranted. Trials of low doses of IL-2 (36,000 to 500,000 U) administered subcutaneously daily or twice daily to HIV-infected patients with CD4+ counts greater than 200 cells/txl resulted in increases in lymphocyte-proliferative responses, NK cell number and activity, and enhanced delayed-type hypersensitivity responses. At the higher doses, modest but transient increases in CD4+ cell counts were also s e e n . 33-35 Higher doses of IL-2 (6 MU to 18 MU daily) have been given intravenously in 5-day cycles every 8 weeks to HIV-infected patients with CD4+ cell counts greater than 200 cells/p.l. 36'37 In these trials, more than half of the patients experienced a substantial polyclonal increase in circulating CD4+ cell counts. This was associated with transient bursts in circulating HIV-1 RNA levels. More recently, subcutaneous administration of IL-2 twice daily for 5 days every 8 weeks in doses up to 15 MU/day were also shown to provide significant increases in circulating CD4+ lymphocyte numbers. 38 As in the continuous intravenous infusion trials, plasma HIV RNA levels increased transiently in the peri-infusion period. Fever, rash, capillary leak, and increased serum transaminases are common toxicities of high-dose IL-2 administration. As plasma levels of TNF-oL rise during IL-2 infusions, this clinical syndrome and the transient increases in viral load have been attributed to induction of TNF-a expression by IL-2. However, neither thalidomide nor chimeric murine-human monoclonal anti-TNF-a antibodies prevented the clinical toxicities or transient increases in plasma HIV RNA levels seen during IL-2 infusions. 39 The clinical significance of the increases in CD4+ T-lymphocyte numbers seen after IL-2 administration remains to be determined. Despite often dramatic increases in circulating CD4+ T-lymphocyte counts, prolonged therapy with IL-2 did not correct the perturbed T-cell receptor VB repertoire seen in HIV-1 disease, s A clinical endpoint trial will be needed to determine the clinical significance of the CD4+ cell increases seen after IL-2 infusions. Design of such a trial will be challenging, since persons with lower CD4+ cell counts, who are at greatest risk of opportunistic infection, are less likely to experience an increase in CD4+ T-cell counts in response to IL-2, whereas clinical endpoints are rare among persons with CD4+ T-cell counts in higher ranges, who are more likely to respond to IL-2. The

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AIDS Clinical Trials Group is now testing the effects of IL-2 among patients with fewer than 350 CD4+ T cells/~l who also are receiving a H A A R T regimen. If H A A R T administration permits a CD4+-cell response to IL-2, a clinical endpoint trial in this population may be more feasible. Interleukin-12. Cells of the monocyte-macrophage lineage are the usual source of IL-12. IL-12 promotes the development of T-helper responses through induction of interferon--y (IFN-~), and may serve as a link between innate and adaptive resistance. 4° Peripheral blood cells obtained from HIVinfected patients produce less IL-12 than do cells of healthy controls, 28'41 but in vivo, IL-12 production in response to endotoxin administration is not impaired in HIV-infected patients. 4z In vitro, IL-12 increases NK-cell cytotoxicity43 and lymphocyte proliferative responses to HIV- and non-HIV-related antigens, 44'4s and can also block programmed cell death in cells obtained from HIV-infected subjects. 46 As is seen with other immune-based therapies, these in vitro effects are more prominent among persons with more preserved immune responses (higher CD4+ T-cell counts). In a recently reported phase I trial, administration of IL-12 at doses of 30 to 1000 ng/kg to HIVinfected subjects with 100 to 500 CD4+ cetls/txl induced a transient decrease in absolute lymphocyte count, and decreased both NK cytotoxicity and antibody-dependent cellular cytotoxicity.47 Ongoing studies are evaluating the immunologic and virologic effects of IL-12 administration to patients with both early and advanced HIV disease. At this point, neither the laboratory nor clinical benefit of this agent are known. Inhibition of proinflammatory cytokine expression. Inhibition of proinflammatory cytokine expression may decrease the immune-activation state that characterizes HIV-1 infection, and also may indirectly inhibit HIV-1 expression. The proinflammatory cytokines TNF-o~ and IL-6 can increase HIV-1 replication, and TNF-o~ may play a role in different manifestations of HIV-1 disease, such as wasting, 4s HIV encephalopathy, 49'5° anemia, 51'52 and hypertriglyceridemia. 53 On this basis, inhibitors of these cytokines have been given to persons with HIV-1 infection to decrease plasma HIV-1 levels or to treat clinical manifestations of HIV-1 infection attributed to overexpression of these cytokines. Tumor neerosisfactor-~. Plasma TNF-oL levels and levels of its soluble receptors are increased in HIVinfected patients, and are predictors of HIV-1 disease progression. 54 TNF-~ can activate HIV-1 expression both in vitro and in vivo, 55'56 and in vitro,

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HIV replication can induce TNF-oL production. 57 In persons with HIV-1 infection, plasma TNFo~ levels correlate with plasma HIV-RNA levels. 58 It is not clear, however, whether in persons with HIV disease TNF-oL drives HIV-1 production, HIV-1 induces overexpression of TNF-a, or the relationship is bidirectional. Thalidomide and pentoxifylline are weak inhibitots of TNF-a production. In HIV-infected patients with no opportunistic infections and CD4+ cell counts above 400 cells/txl, thalidomide at a dose of 100 rag. daily produced no changes in ex vivo c3,tokine production. 59 Addition of thalidomide to antituberculous therapy in the setting of HIV-tuberculosis coinfection decreased plasma TNF-oL and HIVRNA levels. 6° Most recently, thalidomide has been proven an effective treatment for idiopathic oral aphthous ulcers in HIV-infected patients, 61'62 although it is not clear that the treatment effect was related to TNF-~ inhibition, since plasma levels of TNF-e~ and HIV-1 RNA actually rose in the thalidomide-treated patients. 62 Pentoxifylline was found to modestly decrease TNF-oL mRNA and ex vivo TNF-eL production in persons with advanced HIV-1 infection, but plasma viral load was unaffected. 63'64 However, in patients coinfected with HIV-1 and Mycobacterium tuberculosis, administration of pentoxifylline decreased plasma HIV-RNA levels, increased hemoglobin levels, and nonsignificantly reduced TNF-oL production by peripheral blood cells. 65 Thus, thalidomide is a useful therapeutic agent for HIV-associated aphthous ulcers, and weak inhibitors of TNF-a production such as thalidomide and pentoxifylline may have modest effects on HIV-RNA levels in conditions associated with sustained high-level expression of TNF-a, such as tuberculosis. However, these agents do not appear to provide benefit in persons with stable HIV-1 disease. Whether more potent TNF inhibitors, such as corticosteroids, will provide benefit in persons with HIV-1 disease remains to be seen. Interleukin-6. IL-6, together with IL-1 and TNF-~, co-stimulates T-cell proliferation 66 and also upregulates HIV-1 expression. IL-6 may also act as a growth factor for Kaposi's sarcoma and immunoblastic lymphoma cells. 67 As was seen with TNF-oL, plasma IL-6 levels correlate with plasma levels of HIV-1 RNA. 5s When given to patients with AIDS and immunoblastic lymphoma, a murine monoclonal anti-IL-6 antibody produced tumor-growth stabilization, weight gain, and fever resolution. 6s'69 Administration of Tenidap, a nonsteroidal antinflammatory agent that blocks IL-6 production, pro-

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duced no changes in plasma IL-6 or HIV-RNA levels, but increased plasma TNF-oL levels, 5a suggesting that IL-6 expression may negatively regulate plasma levels of TNF-o< At present, the role of IL-6 inhibition in treatment of HIV-1 infection is uncertain. Replacement of cells. Replacement of immunologically active cells or their precursors may help restore depressed immune defenses in persons with AIDS. Potent combinations of antiretroviral agents can successfully decrease plasma levels of HIV-1 to less than the present limits of detection. 9 However, antiretroviral therapies have not eliminated HIVrelated immunologic abnormalities, such as alterations in CD4+-cell function and number, cytokine expression, and thymocyte function, 7° and loss of lymph-node architecture. 71'72 Trials of novel therapeutic strategies, such as adoptive cellular therapies using progenitor cells, CD4+ T cells, or HIV-specific CD8+ CTL, will ascertain whether these approaches can be used to help reconstitute host defenses in persons with HIV-l-induced immune deficiency. CD8+ cell expansion. Cellular defenses against HIV-1 may include MHC-restricted killing of HIV by CTLs, secretion of soluble factors such as CAF that inhibits HIV-1 transcription, 73 or secretion of 13 chemokines that block HIV binding to its co-receptors. 74 CTLs that specifically lyse cells expressing a variety of HIV peptides have been detected. 75-77 The development of a brisk CTL response predicts lower levels of HIV in blood after seroconversion, 2'7s and loss of both CD8+ CTL and CAF activity correlates with disease progression. 79 Thus, expansion and reinfusion of autologous anti-HIVspecific CD8+ CTLs to HIV-1- infected patients may enhance host defenses against HIV. Infusion of autologous activated and expanded CD8+ T cells is well tolerated in HIV disease, but trials of this approach have not been associated with good evidence of either clinical or laboratory benefit attributable to the infused cells. 8°82 Immunization of an HIV-l-seronegative subject, and transfer of anti-gpl60 CTLs to his HIV-l-infected identical twin, resulted in a slight transient increase in CD4+ cell counts, a decrease in 13a-microglobulin, an increased proliferative response to HIV antigen, and activation of C D 8 + / D R + cells. 83 It is unclear whether some of these transient effects were due to activation of the recipient's cells or were related to the passive transfer of specific immunity to HIV-1. Several groups have examined the effects of infusing expanded lines or clones of autologous HIVspecific CD8+ cells. These infusions have not yet

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provided clear evidence of virologic or immunologic benefit. 8°-82'a4 In one of these studies, as many as 20% of viruses isolated after infusion of a nef-specific CTL clone contained mutations in the epitope recognized by the CTL clone. 85 This finding underscores the ability of HIV-1 to evade antiviral strategies, whether immunologic or pharmacologic. Nonetheless, the rapid emergence of mutants after therapy suggests that the infusions resulted in a selection pressure, thus indicating some antiviral activity. The usefulness of the foregoing approach may therefore be limited by the emergence of escape mutations. This in turn may be related to a failure to sustain CTL activity related to inadequate production by CD4+ T cells of helper cytokines for T cells. Co-administration of IL-2 s2 or re-engineering of the IL-2 receptor to permit IL-2-independent CTL proliferation 86 may enhance the durability of CTL activity in this setting. In addition, expansion and infusion of numerous clones with broader specificities may permit a more durable response. CD4+ cell expansion. As stated earlier, the hallmark of HIV infection is a progressive depletion of CD4+ T lymphocytes. Simple replacement of CD4+ T cells after ex vivo expansion provides no assurance that these cells will not be susceptible to HIV-1 infection. Transduction of CD4+ cells with resistance constructs may render them relatively resistant to HIV infection. Recently, CD4+ T cells transduced ex vivo with a rev M10 construct (a transdominant inhibitor of the rev protein that facilitates nuclear export of large viral RNAs) and reinfused into an HIV-l-infected patient tended to survive longer than CD4+ T cells transduced with a control construct. 87 A number of other promising genetic resistance strategies are being developed with the intent of rendering CD4+ T cells or progenitor cells resistant to lytic viral infection in vivo. It should be noted that expression of heterologous genes in adoptively transferred cells may evolve host-immune responses to the relevant gene products, resulting in the elimination of the infused cells. 8s Another interesting approach to CD4+ T cell expansion involves stimulation by immobilized antiCD3 and anti-CD28 antibodies. Expansion by this technique progressively depletes HIV-1 from cultures in vitro, s9 perhaps through enhanced production of chemokines that compete with HIV for fusion co-receptors utilized by M-tropic viruses, s9 as well as by down-modulation of CCR-5 transcripts. 9° Trials of infusion of these expanded CD4+ cell populations are ongoing. The durability of the rela-

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tive resistance to HIV of these cells is unknown. Also unknown is whether infusion of these modified cells will promote the emergence of SI variants of HIV-1 that utilize other co-receptors, such as CCR-3, CXCR-4, or CCR-2, to facilitate cellular infection. Progenitor cell therapies. Transduction of pluripotent hematopoetic cells with viral resistance constructs may promote the generation of T cells and monocytes/macrophages that are relatively resistant to HIV infection. Progenitor cells expressing CD34 can be mobilized into peripheral blood by administration of granulocyte colony-stimulating factor (GCSF), 91'92 harvested, and transduced with HIV-resistance congtructs, such as ribozymes, RNA decoys, transdominant inhibitors of Tat or of Rev, or singlechain antibodies with antiviral activity.93 Potential limitations to the effectiveness of these therapeutic approaches include the low transduction efficiency of retroviral vectors used to deliver the genetic constructs into the progenitor cells, 94'95 the magnitude of gene expression in matured cells, the ability of transduced progenitor cells to undergo maturation in vivo, the level of protection conferred by the constructs, and the functional capacity of the thymus in HIV-infected adults to support T-cell maturation and development. As noted earlier, transduction of ceils with constructs that result in expression of new proteins may lead to host CTL responses against the newly expressed foreign peptides, ss Nonetheless, the ability to insert a genetic marker into progenitor cells provides not only a potential therapeutic avenue in HIV disease, but also a means to track and define the dynamics of cell growth and survival in persons with HIV infection and AIDS. In summary, although these approaches are in early stages of development, the studies in which they are being investigated will add significantly to the understanding of immune ontogeny and of the pathogenesis of immune deficiency in HIV-1 disease. Well-designed trials of host-directed or immunebased therapies can not only examine the utility of a treatment intervention, but can also provide a unique opportunity to test critical hypotheses of HIV disease pathogenesis. Trials of immune-based therapies provide an extraordinary opportunity to expand on observations made in laboratory studies, studies of animal models of HIV-1 disease, or epidemiologic studies. Careful monitoring of the effects of an antiretroviral or immune-based intervention in the context of an HIV-1 treatment trial can not only test the utility of a potential treatment for HIV-1 infection, but can also confirm the relevance of other research findings in the very best model of

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HIV-1 disease, the human being with HIV-1 infection. For example, the failure of soluble CD4 to inhibit HIV replication in vivo despite activity in vitro 96 is a reflection of the weak antiviral activity of this reagent against clinical HIV-1 isolates as opposed to its greater potency against laboratory strains. On the other hand, a convincing demonstration that inhibition of proinflammatory cytokines can block HIV-1 expression can establish the role of this pathway in HIV-1 propagation. As mentioned previously, studies of the feasibility of introducing viral- resistance constructs into progenitor cells can not only confirm the antiviral activity of the construct, but can also provide a mechanism for examining cellular development and immune ontogeny in HIV-1 infection. Appropriate design of immunebased therapeutic trials can maximize the yield of these studies and accelerate the understanding both of useful therapeutic interventions and of the pathogenesis of immune deficiency in AIDS.

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