R. Pat Bucy

Persistence of episomal HIV-1 infection intermediates in patients on highly active anti-retroviral therapy

Treatment of HIV-1-infected individuals with a combination of anti-retroviral agents results in sustained suppression of HIV-1 replication, as evidenced by a reduction in plasma viral RNA to levels below the limit of detection of available assays. However, even in patients whose plasma viral RNA levels have been suppressed to below detectable levels for up to 30 months, replication-competent virus can routinely be recovered from patient peripheral blood mononuclear cells and from semen. A reservoir of latently infected cells established early in infection may be involved in the maintenance of viral persistence despite highly active anti-retroviral therapy. However, whether virus replication persists in such patients is unknown. HIV-1 cDNA episomes are labile products of virus infection and indicative of recent infection events. Using episome-specific PCR, we demonstrate here ongoing virus replication in a large percentage of infected individuals on highly active anti-retroviral therapy, despite sustained undetectable levels of plasma viral RNA. The presence of a reservoir of ‘covert’ virus replication in patients on highly active anti-retroviral therapy has important implications for the clinical management of HIV-1-infected individuals and for the development of virus eradication strategies.

Initial increase in blood CD4+ lymphocytes after HIV antiretroviral therapy reflects redistribution from lymphoid tissues

Previous studies proposed a dynamic, steady-state relationship between HIV-mediated cell killing and T-cell proliferation, whereby highly active antiretroviral therapy (HAART) blocks viral replication and tips the balance toward CD4(+) cell repopulation. In this report, we have analyzed blood and lymph node tissues obtained concurrently from HIV-infected patients before and after initiation of HAART. Activated T cells were significantly more frequent in lymph node tissue compared with blood at both time points. Ten weeks after HAART, the absolute number of lymphocytes per excised lymph node decreased, whereas the number of lymphocytes in the blood tended to increase. The relative proportions of lymphoid subsets were not significantly changed in tissue or blood by HAART. The expression levels of mRNA for several proinflammatory cytokines (IFN-gamma, IL-1beta, IL-6, and macrophage inflammatory protein-1alpha) were lower after HAART. After therapy, the expression of VCAM-1 and ICAM-1 -- adhesion molecules known to mediate lymphocyte sequestration in lymphoid tissue -- was also dramatically reduced. These data provide evidence suggesting that initial increases in blood CD4(+) cell counts on HAART are due to redistribution and that this redistribution is mediated by resolution of the immune activation that had sequestered T cells within lymphoid tissues.

Conferring indirect allospecificity on CD4+CD25+ Tregs by TCR gene transfer favors transplantation tolerance in mice

T cell responses to MHC-mismatched transplants can be mediated via direct recognition of allogeneic MHC molecules on the cells of the transplant or via recognition of allogeneic peptides presented on the surface of recipient APCs in recipient MHC molecules - a process known as indirect recognition. As CD4(+)CD25(+) Tregs play an important role in regulating alloresponses, we investigated whether mouse Tregs specific for allogeneic MHC molecules could be generated in vitro and could promote transplantation tolerance in immunocompetent recipient mice. Tregs able to directly recognize allogeneic MHC class II molecules (dTregs) were obtained by stimulating CD4(+)CD25(+) cells from C57BL/6 mice (H-2(b)) with allogeneic DCs from BALB/c mice (H-2(d)). To generate Tregs that indirectly recognized allogeneic MHC class II molecules, dTregs were retrovirally transduced with TCR genes conferring specificity for H-2K(d) presented by H-2A(b) MHC class II molecules. The dual direct and indirect allospecificity of the TCR-transduced Tregs was confirmed in vitro. In mice, TCR-transduced Tregs, but not dTregs, induced long-term survival of partially MHC-mismatched heart grafts when combined with short-term adjunctive immunosuppression. Further, although dTregs were only slightly less effective than TCR-transduced Tregs at inducing long-term survival of fully MHC-mismatched heart grafts, histologic analysis of long-surviving hearts demonstrated marked superiority of the TCR-transduced Tregs. Thus, Tregs specific for allogeneic MHC class II molecules are effective in promoting transplantation tolerance in mice, which suggests that such cells have clinical potential.

Avian T cell ontogeny

Publisher Summary This chapter focusses on avian T cell development. A renewed interest in avian T cell development has emerged with the use of monoclonal and functional antibodies to elucidate T cell differentiation antigens and molecular and functional definitions of mammalian T cell receptors (TCRs). Thousands of avian species have been identified; the domestic chicken has served as the avian representative in most studies of the immune system. Chick-quail chimeras have also proved to be an especially informative model. The chapter focuses on information gained in studies of these model systems. Monoclonal antibodies are produced against a variety of functionally important molecules expressed on the surface of chicken T cells and most of these have well-defined mammalian counterparts. The chicken T cell receptors can be divided into three groups, each of which is recognized by a different monoclonal antibody. Direct evidence for thymus-directed chemotaxis of hematopoietic precursors is obtained by observing migration of cells toward the thymic epithelium in an in vitro model. Comparative analysis of avian T cell development and function reveals striking conservation of the major features defined for the mammalian T cell system. In both mammals and birds, the T cells utilize either γ/δTCR or α/βTCR together with a CD3 protein complex as signal-transducing receptors for antigen presented in the context of major histocompatibility complex restriction elements.

Quantitation of HIV-1 by real-time PCR with a unique fluorogenic probe.

Quantitation of HIV-1 specific RNA and DNA is pivotal to understanding the pathophysiology of HIV-1 diseases. A method has been developed for quantitation of HIV-1 DNA/RNA by real-time PCR using a unique fluorogenic primer-probe adduct known as scorpion. The probe hybridises to the extension of the adjoining primer intramolecularly, a process kinetically and thermodynamically more favourable than the conventional bimolecular probe-target hybridisation. Data presented in this paper indicate that the scorpion assay is extremely robust and is quite comparable to beacon-based assays. The scorpion assay is also comparable to quantitative competitive PCR (QC--PCR) assays but requires only a fraction of time and effort. Additionally, the dynamic range of the scorpion assay is several log-fold higher than the conventional end point PCR assays. As few as ten copies of vDNA can be detected in the presence of a large excess of exogenously added genomic DNA. Limiting dilution analysis indicates that the assay is capable of detecting a single copy of the viral template. Thus, the scorpion assay presents a specific and sensitive approach for quantitation of DNA/RNA templates by real-time PCR.

Recurrence of the Acute HIV Syndrome after Interruption of Antiretroviral Therapy in a Patient with Chronic HIV Infection: A Case Report

Acute HIV infection frequently results in a syndrome characterized by high fevers, headache, erythematous rash, myalgia, and adenopathy concurrent with extremely high plasma HIV RNA levels, often more than 1 000 000 copies/mL (1, 2). Anecdotal experience suggests that acute HIV symptoms improve with potent antiviral therapy but may recur if therapy is not sustained over several weeks (3). Little is known about the potential consequences of temporary interruptions in therapy, either in cases of acute seroconversion illness or in the more common clinical setting of established HIV infection. Scheduled treatment interruption is being explored as a component of immune-based therapeutic strategies for HIV infection (4), but the long-term risks and benefits of this approach are uncertain. We recently completed a pilot study to evaluate the effects and safety of a single 8-day interruption of highly active antiretroviral therapy in chronically infected, successfully treated patients. Since the conception of this study, other reports have suggested that incomplete or intermittent viral load suppression in selected patients with primary HIV infection may stimulate effective HIV-specific cellular immune responses and allow persistent immune control after therapy is stopped (5-7). It is not clear whether similar results will be seen in patients initiating therapy long after primary HIV infection. The risks and benefits of this approach are likely to be different in later stages of HIV infection than in acute HIV infection. We describe the evaluation of a febrile syndrome that developed when one of the previous participants in our pilot study again self-discontinued therapy. Plasma viral load assays were performed by using standard methods (Amplicor, Roche, Somerville, New Jersey). Flow cytometry for T-cell subset analysis was performed by using standard methods certified by the AIDS Clinical Trials Group. The funding sources had no role in gathering, analyzing, or interpreting the data or in the decision to submit the paper for publication. Case Report The patient, a 30-year-old man, began taking antiretroviral therapy (zidovudine, 300 mg twice daily; lamivudine, 150 mg twice daily; and indinavir sulfate, 800 mg three times daily) approximately 3 months after a high-risk HIV exposure event that was followed by a self-limited mononucleosis-like illness. His pretherapy plasma HIV RNA level was 880 000 copies/mL. While he maintained this antiretroviral regimen (>2 years), his viral load remained less than 50 copies/mL and his absolute CD4 cell count increased from a nadir of 0.085 109 cells/L to sustained levels of more than 0.500 109 cells/L. The patient temporarily interrupted antiretroviral therapy more than 2 years later as part of an investigational pilot study described elsewhere (Kilby and colleagues. In preparation). Over the 8-day scheduled treatment interruption, his viral load increased from less than 50 copies/mL to 1921 copies/mL and his absolute CD4 count decreased from 0.501 109 cells/L to 0.375 109 cells/L. After the patient resumed the same antiretroviral regimen, his viral load was again repeatedly less than 50 copies/mL, except for two isolated minimal elevations (77 copies/mL and 737 copies/mL). His CD4 count consistently remained above 0.500 109 cells/L (Figure). Figure. Changes in plasma viral load ( squares ) and absolute CD4 cell count ( circles ) over time in a patient with chronic HIV infection. The patient returned for a regular clinic visit 169 days after the scheduled treatment interruption. At this visit, he was asymptomatic, the CD4 count was 0.743 109 cells/L, the leukocyte count was 9.1 109 cells/L, the hematocrit was 0.49, the platelet count was 195 109 cells/L, and the viral load was less than 50 copies/mL. The patient received a routine influenza vaccination. On the same day, without informing caregivers, he chose to interrupt his antiretroviral regimen because of concerns about impending insurance problems. He returned 11 days later with fever (body temperature, 39.4 C) and reported malaise, tender adenopathy, myalgia, emesis, and loose stools. He stated that his symptoms were identical to but more severe than those experienced during his episode of presumed acute HIV syndrome 4 years previously. He initially said that he had not discontinued therapy and was treated symptomatically with antipyretics and analgesics. The patient returned for follow-up after 14 days without therapy and showed no improvement. Laboratory analysis revealed a CD4 count of 0.164 109 cells/L, a leukocyte count of 3.2 109 cells/L, a platelet count of 68 109 cells/L, and a viral load of 327 874 copies/mL. He was admitted to the hospital because he had had a fever (body temperature>39 C) for more than 10 days and had additional symptoms of pharyngitis and a transient erythematous truncal rash. On the 21st day after interruption of therapy, the patient informed physicians that he had stopped therapy immediately after receiving the influenza vaccination. At this time, his viral load was more than 1 000 000 copies/mL and his CD4 count was 0.086 109 cells/L. Physical examination was notable only for a transient erythematous rash on the torso and tender cervical and inguinal adenopathy. Routine and lysis centrifugation blood cultures for mycobacteria and fungi were negative. Acute and convalescent serologic tests showed no evidence of acute infection with cytomegalovirus, EpsteinBarr virus, human herpesvirus 6, parvovirus, rubeola, or rubella. A tuberculin skin test was nonreactive. Antigen assays and serologic tests for cryptococcosis, histoplasmosis, and coccidioidomycosis were also negative. Routine throat culture was negative. An excisional cervical lymph node biopsy revealed preserved architecture but marked depletion of paracortical T cells. Flow cytometry showed no evidence of a clonal lymphoproliferative process. Special stains for mycobacterial and fungal pathogens were negative. Immunoperoxidase reactions for cytomegalovirus and EpsteinBarr virus were negative, and HIV p24 antigen staining was positive. When the same antiretroviral regimen was reinstituted on the 21st day, the patients fever and other symptoms rapidly resolved. In less than 2 weeks, his viral load decreased to 2743 copies/mL and his absolute CD4 count increased to more than 0.500 109 cells/L. Two months later, his viral load was 59 copies/mL and his absolute CD4 count was 0.511 109 cells/L. Discussion Our report describes recapitulation of the acute retroviral syndrome in a patient with chronic HIV infection. This syndrome was associated with profound viral rebound that followed simultaneous administration of influenza vaccination and interruption of HIV therapy. The dynamic increase in plasma HIV RNA level after discontinuation of therapy coincided with a prolonged febrile illness indistinguishable from the patients primary seroconversion symptoms. This case suggests that relapses of the acute retroviral syndrome, described in a recent report involving therapeutic interruption during acute HIV seroconversion (3), may also occur in persons who have undergone successful virologic suppression for years after primary HIV infection. It is not clear whether the patients history of a brief scheduled treatment interruption had any effect on the results of the second interruption. Although nonspecific immune activation resulting from a brief increase in viremia could provoke the acute HIV syndrome during subsequent interruptions, this would seem to be an unlikely explanation for events occurring after 6 more months of potent viral suppression. The temporal relationship between these acute symptoms and influenza vaccination may be merely coincidental. However, it is conceivable that vaccination within hours of treatment interruption could have escalated the viral rebound syndrome. In the absence of potent combination therapy, influenza vaccinations (8) as well as other routine immunizations (9, 10) have been shown to transiently activate HIV replication; however, this phenomenon is generally attenuated and is not associated with symptoms in patients receiving potent antiretroviral regimens (11, 12). In our patient, the dramatic rate of change in plasma viral load suggests that the extraordinarily high level of HIV replication, de novo infection, and cellular activation probably played a role in the development of symptoms. It is notable that the patient had not been symptomatic when he presented with a similarly high viral load before initiation of treatment 4 years previously. The brisk, dramatic shifts in absolute CD4 count on and off therapy in this case may reflect redistribution of cells from inflamed tissues to blood rather than production of new T cells, as previously described (13, 14). Carefully designed investigations involving immune-based strategies, including scheduled treatment interruption and therapeutic immunization, seem warranted. However, our report reinforces the importance of diligent, frequent monitoring of patients with chronic HIV infection whenever effective antiretroviral therapy is abruptly discontinued.

A randomized therapeutic vaccine trial of canarypox-HIV-pulsed dendritic cells vs. canarypox-HIV alone in HIV-1-infected patients on antiretroviral therapy

Targeting canarypox (CP)-HIV vaccine to dendritic cells (DCs) elicits anti-HIV-1 immune responses in vitro. We conducted a phase I/II clinical trial to evaluate whether adding DC to a CP-HIV vaccine improved virologic control during analytic treatment interruption (ATI) in HIV-1-infected subjects. Twenty-nine subjects on suppressive antiretroviral therapy were randomized to vaccination with autologous DCs infected with CP-HIV+keyhole limpet hemocyanin (KLH) (arm A, n=14) or CP-HIV+KLH alone (arm B, n=15). The mean viral load (VL) setpoint during ATI did not differ between subjects in arms A and B. A higher percentage of subjects in the DC group had a VL setpoint < 5,000 c/mL during ATI (4/13 or 31% in arm A compared with 0/13 in arm B, p=0.096), but virologic control was transient. Subjects in arm A had a greater increase in KLH lymphoproliferative response than subjects in arm B; however, summed ELISPOT responses to HIV-1 antigens did not differ by treatment arm. We conclude that a DC-CP-HIV vaccine is well-tolerated in HIV-1-infected patients, but does not lower VL setpoint during ATI compared with CP-HIV alone. New methods to enhance the immunogenicity and antiviral efficacy of DC-based vaccines for HIV-1 infection are needed.

Evidence That Intermittent Structured Treatment Interruption, but Not Immunization with ALVAC-HIV vCP1452, Promotes Host Control of HIV Replication: The Results of AIDS Clinical Trials Group 5068

BACKGROUND The ability to control human immunodeficiency virus (HIV) replication in vivo in the absence of antiretroviral therapy (ART) is a measure of the efficiency of antiviral immunity. In a study of patients with chronic, ART-suppressed HIV infection, AIDS Clinical Trials Group 5068 investigated the effects of immunization with an exogenous HIV vaccine and pulse exposure to the subjects unique viral epitopes, by means of structured treatment interruptions (STIs), on the dynamics of viral rebound during a subsequent analytical treatment interruption (ATI). METHODS Ninety-seven subjects receiving stable ART with an HIV-1 RNA load <50 copies/mL and CD4(+) T lymphocyte count >400 cells/mm(3) were randomized to undergo continued ART, STIs, ALVAC-HIV vCP1452 immunization, or STIs and ALVAC-HIV vCP1452 immunization. RESULTS Subjects in the 2 STI arms had a significantly longer median doubling time in the period of the initial rise of viral load, a significantly lower median peak viral load, a significantly lower median end-of-ATI viral load set point, and a greater proportion of subjects with an end-of-ATI viral load set point <1,000 copies/mL, compared with the subjects in the 2 arms without STIs. With an immunization schedule of 3 sets of 3 weekly injections, ALVAC-HIV vCP1452 did not affect viral load measures. CONCLUSIONS In this randomized, controlled study of intermittent STI as a therapeutic autoimmunization strategy, evidence of enhanced immunologic control of HIV replication was demonstrated.

The Arthus Reaction in Rodents: Species-Specific Requirement of Complement

We induced reverse passive Arthus (RPA) reactions in the skin of rodents and found that the contribution of complement to immune complex-mediated inflammation is species specific. Complement was found to be necessary in rats and guinea pigs but not in C57BL/6J mice. In rats, within 4 h after initiation of an RPA reaction, serum alternative pathway hemolytic titers decreased significantly below basal levels, whereas classical pathway titers were unchanged. Thus the dermal reaction proceeds coincident with systemic activation of complement. The serine protease inhibitor BCX 1470, which blocks the esterolytic and hemolytic activities of the complement enzymes Cls and factor D in vitro, also blocked development of RPA-induced edema in the rat. These data support the proposal that complement-mediated processes are of major importance in the Arthus reaction in rats and guinea pigs, and suggest that BCX 1470 will be useful as an anti-inflammatory agent in diseases where complement activation is known to be detrimental.

A Randomized, Partially Blinded Phase 2 Trial of Antiretroviral Therapy, HIV‐Specific Immunizations, and Interleukin‐2 Cycles to Promote Efficient Control of Viral Replication (ACTG A5024)

Strategies to limit life-long dependence on antiretroviral therapy (ART) are needed. We randomized 81 human immunodeficiency virus (HIV)-infected subjects to 4 interventional arms involving continued ART plus ALVAC vCP1452 (or placebo) with or without interleukin (IL)-2 infusions. Viral load rebound 12 weeks after ART interruption was then analyzed to assess immune control. Fifty-two subjects reached the study end point. ALVAC recipients had 0.5 log(10) lower virologic rebounds (P=.033). IL-2 plus vaccine boosted CD4(+) T cell counts (P<.001) but did not diminish viral rebound. Significant changes were not detected for HIV-specific lymphoproliferative responses in any arm. This exploratory protocol provides useful clinical data for future therapeutic immunization trial design.