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CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the Unit of Human Virology, AIDS
Immunopathogenesis Unit, DIBIT, and Clinic of Infectious Disease, San
Raffaele Scientific Institute, Milan, Italy; Department of Public
Health and Cellular Biology, University Tor Vergata, Rome, Italy;
Department of Pediatrics, University of Milan, Italy; and Laboratory of
Virology, Istituto Superiore di Sanita, Rome, Italy,
The combination of interleukin 2 (IL-2) and antiretroviral
therapy (ART) represents an emerging strategy in the treatment of
patients infected with HIV. Aside from its immunomodulatory role,
however, IL-2 may induce replication of human herpesvirus 8 (HHV-8)/Kaposi sarcoma (KS)-associated herpesvirus. We retrospectively evaluated HHV-8 plasma viremia and cellular load, as well as
anti-HHV-8 antibody titers, in sequential samples from 84 patients
receiving ART alone or in combination with IL-2. At baseline, HHV-8
plasma viremia was present only in 2 HHV-8-seropositive patients in
whom KS subsequently developed during or immediately after termination of IL-2 therapy. The level of viremia increased during follow-up and
peaked at the time of the clinical manifestation of KS. Moreover, transient peaks of HHV-8 viremia were temporally associated with administration of IL-2. HHV-8 plasma viremia was never detected in the
other 47 patients receiving IL-2 nor in 35 controls treated only with
ART. Thus, IL-2 therapy seems safe in most patients infected with both
HIV and HHV-8, except for those with detectable HHV-8 viremia, who may
not be eligible for IL-2 treatment.
(Blood. 2002;100:1575-1578) Although therapeutic regimens that combine low
doses of interleukin 2 (IL-2) with antiretroviral therapy (ART) have
been used without such major clinical concerns as high-grade toxicity
or increased levels of HIV-1 viremia,1,2 the effects of
IL-2 on replication of human herpesvirus 8 (HHV-8)/Kaposi sarcoma
(KS)-associated herpesvirus and the development of KS, which is
20 000-fold more frequent in patients with AIDS than in the general
population,3 must be evaluated carefully. In vitro, some
IL-2-induced cytokines, such as IL-1 Because of the high seroprevalence of HHV-8 in the Italian
population,8-11 particularly among patients with HIV
infection,12,13 we assessed the effect of IL-2 therapeutic
regimens on HHV-8 plasma and cellular load and correlated the latter
with the development of KS. For this purpose, we investigated
sequential plasma samples and peripheral blood mononuclear cell (PBMC)
pellets from a cohort of HIV-1-infected patients enrolled in a
randomized phase 2 trial of administration of IL-2 with ART. To provide
a control, we studied a cohort of patients with recent HIV
seroconversion who were treated with the same ART protocol and followed
for a comparable length of time.
To measure HHV-8 DNA load in biologic samples, we developed a novel
real-time quantitative polymerase chain reaction (PCR) system that
combines TaqMan technology with use of a synthetic DNA calibrator
molecule to normalize the intersample extraction recovery and monitor
PCR artifacts. Using this new assay, we demonstrated, for the first
time, a strict temporal correlation between multiple administrations of
IL-2 and increases in HHV-8 viremia in patients with a detectable HHV-8
plasma load before the beginning of IL-2 administration.
Patients and sample selection
Quantification of HHV-8 DNA by real-time quantitative PCR
Serologic assays A total of 170 plasma samples obtained from 84 HIV-infected patients were investigated for the presence of antibodies (Abs) against lytic and latent HHV-8 antigens. Abs to lytic antigens of HHV-8 were detected by using an immunofluorescent assay (IFA) based on the BCBL-1 cell line,15 whereas Abs against latent HHV-8 antigens were detected by using an IFA based on the BCP-1 cell line as described previously.16
No major differences were observed in the baseline characteristics
of the 2 groups of patients enrolled in the IL-2 protocol (the group
treated with ART plus IL-2 and the control group treated only with
ART), except for a slightly higher HIV-1 viral load in the
group treated with the combination therapy (Table
1). Not surprisingly, the group of
ART-treated patients with recent HIV-1 seroconversion had higher mean
CD4 cell counts and HIV-1 plasma loads and no intravenous drug users
(Table 1). However, all patient groups were homogeneous with respect to
treatment, age, sex, follow-up, and HHV-8 seroprevalence (Table 1). In
agreement with previous observations,13,17 HHV-8
seroprevalence was remarkably high among homosexual men (21 of 38 [55%]).
HHV-8 DNA was not detectable in any of the samples obtained from 53 HHV-8-seronegative patients (252 plasma and PBMC samples) or from 14 HHV-8-seropositive patients treated with ART (80 plasma and PBMC
samples). Only in 4 patients treated with IL-2 plus ART was HHV-8 DNA
detected in circulating mononuclear cells, and in only 2 of them
(patient 70 [Figure 1B] and patient 72, 12 and 65 HHV-8 genome equivalents/105 cells, respectively)
was it found repeatedly in all tested samples. The results for all
PCR-positive PBMC samples (n = 8) were concordant with the antilytic
but not the antilatent Ab test results. HHV-8 plasma viremia was
detectable only in the same 2 patients (patient 70 [Figure 1A and 1C]
and patient 72, 100 and 80 HHV-8 genome equivalents/mL, respectively),
both of whom had subsequent development of KS (during IL-2 treatment in
patient 72 and 5 months after the end of the 1-year period of IL-2
administration in patient 70). In both patients, HHV-8 viremia was also
present at baseline, before the beginning of IL-2 administration.
Although large cohort studies have demonstrated that HHV-8 seropositivity predicts progression to KS in HIV-positive homosexual men,17-19 only HHV-8 plasma viremia was clearly associated with the development of KS in our cohort. Neither the anti-HHV-8 Ab titer nor the detection of HHV-8 sequences in circulating PBMCs could unambiguously identify patients at risk for subsequent development of KS. To date, none of the other patients seropositive for HHV-8 have had onset of KS, even though some HHV-8-seropositive patients had a second round of IL-2 plus ART. Because both patients in whom KS developed were homosexuals, we calculated the incidence of KS among our homosexual patients (n = 38). The incidence was 11.4 cases/1000 person-years; this did not differ significantly (on 2-tailed Fisher exact test) from the overall incidence of KS observed in Italian HIV-seropositive homosexuals.13 We analyzed the dynamics of HHV-8 plasma viremia and cellular load in one patient (patient 70) for whom a considerable number of sequential samples was available (Figure 1A and 1B). HHV-8 plasma load was low during the first 4 months of observation (57-138 genome equivalents/mL). It then increased significantly during IL-2 therapy, ranging from 219 to 492 genome equivalents/mL, and peaked at the time of the clinical manifestation of KS (2097 genome equivalents/mL). Similarly, the PBMC viral load (Figure 1B) increased from the beginning to the end of the IL-2 therapy (from 180 to 538 genome equivalents/105 cellular genomes), reaching a maximum of 2194 genome equivalents at the time of KS manifestation. The lytic Ab titers measured at the same time points did not vary significantly (Figure 1B). A low but constant level of cellular DNA contamination was observed in all plasma samples (between 210 and 410 cellular genome equivalents/mL), thus ruling out the possibility that the variations in HHV-8 plasma load were due to a different rate of destruction of latently infected cells during manipulation of samples (Figure 1A). We then correlated the degree of IL-2-induced immune reconstitution with the dynamics of HHV-8 viremia by analyzing plasma samples obtained from patient 70 during 3 cycles (the first, third, and last) of IL-2 administration (Figure 1C). Three time points were analyzed in each cycle: baseline (just before the first IL-2 injection), the third day (after 5 injections of IL-2), and the 8th day (2 days after the last IL-2 injection of the cycle). HHV-8 viremia progressed steadily, despite a significant increase in CD4 cell count (from 192 to 722 cells/µL; Figure 1B) and a progressive normalization of the ratio of CD4 to CD8 cells (from 0.26 to 1.02; data not shown). CD4 and CD8 cell counts decreased dramatically on the third day of IL-2 administration. This was followed by a quick recovery of CD8 cells and a sharp increase in the number of circulating CD4 cells (Figure 1C). Interestingly, the HHV-8 viremia had an opposite pattern; it increased with each IL-2 injection and returned quickly to the baseline level shortly after the last IL-2 administration, although the peak of viremia was more prolonged during the last cycle of treatment. The increase in HHV-8 viremia during IL-2 administration was also observed in the few plasma samples available from the other patient who had appearance of KS lesions during IL-2 treatment (baseline, < 10 genome equivalents/mL; third day, 80 genome equivalents/mL; and 8th day, < 10 genome equivalents/mL). Although in vitro IL-2 does not seem to induce HHV-8 replication
in chronically infected, primary effusion lymphoma (PEL)-derived cell
lines,4 IL-2-induced inflammatory cytokines can alone sustain production of HHV-8 viral particles in primary blood-derived mononuclear cells and in a PEL-derived B-cell line,
BCBL-1.4,5 Moreover, the observed temporal
relation between the peaks of HHV-8 viremia and the drastic reduction
in circulating CD4 and CD8 cells suggests that some of the pathological
mechanisms involved in the IL-2-induced vascular leak
syndrome20 may also contribute to the spread of HHV-8
infection and the appearance of KS lesions. Indeed, productively
HHV-8-infected mononuclear cells could adhere to cytokine-activated
endothelial cells21 and propagate the infection to the
microvascular endotelium.22 Alternatively, infected cells
could migrate into the tissue and differentiate into spindlelike
endothelial cells on exposure to IFN- Several epidemiologic studies have reported a direct correlation between low CD4 cell counts and manifestations of KS.12,17,18 In our patients, however, KS occurred despite a progressive reconstitution of CD4 T-cell counts, suggesting that more subtle qualitative defects of the immune system may account for the appearance of KS. Indeed, in these patients, a defective or incomplete restoration of the specific anti-HHV-8 immune response in the context of a more reactive, cytokine-boosted immune system might represent a favorable milieu for the development of KS. In this study, we observed the first evidence that in vivo administration of IL-2 is temporally associated with transient increases in HHV-8 viremia and, more importantly, that persistent HHV-8 replication is required for subsequent development of KS lesions. However, the finding that KS occurred only in patients in whom HHV-8 replication was already detectable (albeit at a lower level) before initiation of the IL-2 regimen suggests that IL-2-based therapeutic regimens can be safely implemented in most patients with both HIV and HHV-8 infection. Indeed, the incidence of KS in the homosexual patients studied was not significantly greater than the overall KS incidence among Italian homosexuals infected with HIV-1. Special caution regarding safe use of IL-2-based approaches is necessary only in patients who have signs of pre-existing active HHV-8 infection or HHV-8-associated disease. Therefore, specific assays for detecting active HHV-8 infection might represent a useful clinical management tool in dually infected patients, especially in populations at higher risk of development of KS.
We thank Dr Priscilla Biswas for critically reviewing the manuscript and Ms. Stefania Laus for editorial assistance.
Submitted August 16, 2001; accepted April 15, 2002.
Supported by grants from the II Italian National AIDS Project, Ministry of Health, Rome.
M.M. and F.B. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Mauro S. Malnati, Unit of Human Virology, Via Olgettina 58, 20132 Milan, Italy; e-mail: malnati.mauro{at}hsr.it.
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© 2002 by The American Society of Hematology.
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  X. Zhang, J. F. Wang, B. Chandran, K. Persaud, B. Pytowski, J. Fingeroth, and J. E. Groopman Kaposi's Sarcoma-associated Herpesvirus Activation of Vascular Endothelial Growth Factor Receptor 3 Alters Endothelial Function and Enhances Infection J. Biol. Chem., July 15, 2005; 280(28): 26216 - 26224. [Abstract] [Full Text] [PDF]
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 F. Broccolo, G. Locatelli, L. Sarmati, S. Piergiovanni, F. Veglia, M. Andreoni, S. Butto, B. Ensoli, P. Lusso, and M. S. Malnati Calibrated Real-Time PCR Assay for Quantitation of Human Herpesvirus 8 DNA in Biological Fluids J. Clin. Microbiol., December 1, 2002; 40(12): 4652 - 4658. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
and interferon-
(IFN-
80°C in at least 2 aliquots that were never
thawed before analysis.
) and 
), HHV-8 load of
PBMCs (
), and anti-HHV-8 lytic antigens Ab titer (
), in patient
70. IL-2 was administered subcutaneously every 8 weeks for a total of 6 cycles (5 days twice a day for each cycle). KS lesions developed after
12 months of IL-2 therapy, 5 months after the last
administration of IL-2. (C) Dynamics of HHV-8 plasma viremia (
) and
CD4 (