Kaposi's sarcoma-associated herpesvirus-specific immune reconstitution and antiviral effect of combined HAART/chemotherapy in HIV clade C-infected individuals with Kaposi's sarcoma
Bihl, Floriana; Mosam, Anisac; Henry, Leah Na; Chisholm, John V IIIa; Dollard, Sheilae; Gumbi, Pamelag; Cassol, Edanai,k; Page, Tarynd; Mueller, Nicolasj; Kiepiela, Photinig; Martin, Jeff Nf; Coovadia, Hoosen Mh; Scadden, David Tb,l; Brander, Christiana
From the aPartners AIDS Research Center, Massachusetts General Hospital and Harvard Medical School, USA
bCenter for Regenerative Medicine and Technology, Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, Massachusetts, USA
cDepartment of Dermatology, South Africa
dHIV Pathogenesis Program, Doris Duke Medical Research Institute, South Africa
eDepartment of Virology, South Africa
fCentre for HIV/AIDS Research, Faculty of Health Science, Nelson R. Mandela School of Medicine, University of KwaZulu-Natal, Durban, South Africa
gCenters for Disease Control and Prevention, Atlanta, Georgia, USA
hAIDS Immunopathogenesis Unit DIBIT, San Raffaele Scientific Institute, Milan, Italy
iDivision of Infectious Diseases and Hospital Epidemiology, University Hospital of Zurich, Zurich, Switzerland
jHIV-1 Immune Pathogenesis and Therapeutics Research Program, University of Pretoria, Pretoria, South Africa
kDepartments of Epidemiology and Biostatistics, University of California, San Francisco, California, USA
lHarvard Stem Cell Institute, Boston, Massachusetts, USA.
Received 10 October, 2006
Revised 7 March, 2007
Accepted 16 March, 2007
Correspondence to Christian Brander, PhD, Partners AIDS Research Center, Building149, 13th Street, Charlestown, MA 02129-2000, USA. Tel: +1 617 724 5789; fax: +1 617 726 5411; e-mail: email@example.com
The first two authors contributed equally to this work.
Background: Kaposi's sarcoma-associated herpesvirus (KSHV) is endemic in South Africa and the clinical manifestation of AIDS-associated Kaposi's sarcoma (KS) represents a significant clinical problem. Whereas the positive effects of HAART on the regression of KS have been well established, less is known about the role of herpesvirus-specific cellular immunity in disease improvement.
Design: Thirty-three treatment-naive HIV clade C-infected individuals with KS were randomly assigned into two treatment arms (HAART plus systemic chemotherapy versus HAART alone). KSHV-specific cellular immune responses, viral loads and clinical outcome were evaluated.
Methods: KSHV, Epstein–Barr virus and HIV-specific cellular immunity was measured using an IFN-γ enzyme-linked immunospot assay in samples obtained at baseline and up to 11 months after treatment initiation. Cell-associated KSHV viremia was determined by real-time polymerase chain reaction.
Results: Robust increases in CD4 cell counts and suppressed HIV viral loads were seen in parallel with significant increases in the KSHV-specific cellular immune responses over time. Although slowly increasing after 5 months, KSHV-specific T-cell responses were significantly elevated only after 11 months, with both lytic and latent antigens being more frequently targeted. A trend towards better clinical outcome with HAART plus chemotherapy treatment was observed compared with HAART alone, and was accompanied by a significant reduction in cellular KSHV viral load in the HAART plus chemotherapy-treated subjects but not those treated with HAART alone after 11 months of treatment.
Conclusion: The data show a temporal association between the clinical improvement of KS and the re-appearance of KSHV-specific cellular immunity, and demonstrate an effective suppression of KSHV viral replication using combination therapy.
Sub-Saharan Africa is severely affected by the global AIDS epidemic, accounting for more than 28 million infected individuals and 2 million deaths per year . The seroprevalence of Kaposi's sarcoma-associated herpesvirus (KSHV, or HHV-8) in South Africa is high, approaching 40% in some populations [2–5]. In the wake of the AIDS epidemic, the clinical manifestations of Kaposi's sarcoma (KS) have increased dramatically and KS has become the most common cancer of men in sub-Saharan Africa [6–9]. The resolution of KS after the initiation of HAART has been well documented in clade B-infected individuals and in countries where HAART has been widely available [10–12]. Furthermore, systemic chemotherapy using classic combination regimens including doxorubicin (adriamycin), bleomycin and vincristin (ABV regimen) or single agent approaches (pegylated liposomal doxorubicin, paclitaxel, or etoposide) has proved effective in the treatment of HIV-associated KS when combined with HAART [13,14]. Despite the clinical importance of KS-associated mortality in sub-Saharan Africa, where HIV and KSHV seroprevalences are both high, only little is known regarding the relative benefits of HAART and chemotherapy for the treatment of KS in clade C-infected patients of non-Caucasian descent and the role the host's immune response plays in the control of KSHV. The present study was designed to assess the KSHV-specific cellular immunity prospectively in individuals with KS and undergoing treatment with either HAART or HAART and chemotherapy. Thirty-three HAART-naive patients with KS lesions were randomly selected for treatment and studied for disease outcome, HIV and KSHV viral loads, and antiviral cellular immunity over the first year after treatment initiation. The data show a more effective suppression of viral replication in individuals receiving combination therapy compared with HAART alone and an overall significant reconstitution of the KSHV-specific cellular immune response, suggesting that the observed regression of KS in HAART-treated individuals might be at least partly immune mediated.
Thirty-three patients with treatment-naive HIV infection and biopsy-confirmed KS lesions were staged according to the TIS classification [assessing the tumor extent (T), the immune system's CD4 cell count (I) and the severity of systemic illnesses (S)] [15,16] into groups of individuals with good and poor prognosis and then randomly assigned to two treatment arms. The treatment arms were either HAART alone, a fixed-dose combination of two generic nucleoside reverse transcriptase inhibitors (stavudine 40 mg; and lamivudine 150 mg) and one non-nucleoside reverse transcriptase inhibitor (nevirapine 200 mg)  or HAART in combination with systemic chemotherapy, consisting of 2-week cycles of the classic ABV regimen including bleomycin (10 U/m2), doxorubicin (20 mg/m2), and vincristine (1.4 mg/m2), starting at day 28 [11,18]. Seventeen patients received HAART alone and 16 HAART in combination with chemotherapy. One female patient was not randomly assigned because of pregnancy and received HAART alone. HIV viral loads and CD4 cell counts were available for all subjects at baseline, 5 months and 11 months. The immunological analyses were performed at baseline and 11 months of therapy and KSHV viral loads were determined at three timepoints (baseline, 5 months and 11 months). As a result of fatal disease outcome and sample availability, the number of subjects assessed virologically and immunologically differed between timepoints as indicated in the figure legends. KS disease at 11 months was staged as previously defined by the AIDS Clinical Trials Group , and subjects were accordingly grouped into either complete responders (n = 10) showing complete regression of all KS lesions, including tumor-associated edema, for at least 4 weeks; partial responders (n = 17) presenting with more than a 50% decrease in the number and size of previous existing lesions for at least 4 weeks and the flattening of nodular lesions to indurated plaques; and progressors (n = 6) presenting with progressively enlarging (> 25% compared with previous lesions) or new lesions or new sites of disease, the development of new or increasing tumor-associated edema or effusion or fatal KS outcome. The Institutional Review Boards of the participating hospitals approved the study and all subjects provided written informed consent before recruitment.
Synthetic Kaposi's sarcoma-associated herpesvirus peptide sets
A total of 309 synthetic overlapping peptides were generated covering the entire K8.1 protein (35 peptides), K10.5 (76 peptides), K12 (51 peptides), open reading frame (ORF)57 (41 peptides), the minor capsid glycoprotein (ORF65; including 57 residues spanning the previously reported C-terminal extended ORF65 sequence; 21 peptides)  and the latency-associated nuclear antigen (LANA or ORF73; 85 peptides). Peptides were synthesized as overlapping 18–20 mers, based on the reported KSHV viral sequence isolated from the KSHV-infected BC-1 cell line . Furthermore, 93 overlapping peptides were included that spanned the HIV clade C Gag and Nef protein sequences as described . In addition, 127 previously defined Epstein–Barr virus (EBV)-derived T-cell epitopes were included to assess EBV-specific T-cell reactivity .
Enzyme-linked immunospot assay
IFN-γ enzyme-linked immunospot (ELISpot) assays were performed as previously described [22,23], using 200 000 thawed peripheral blood mononuclear cells (PBMC)/well for the detection of KSHV-specific responses and 50 000–100 000 PBMC to assess responses to HIV or EBV-encoded antigens. Peptides were added in protein-specific peptide pools, containing each peptide at a final concentration of 10 μg/ml. No peptide was added to four wells serving as negative control wells. Phytohemagluttinin (Remel, Hythe, Kent, UK) was added at a concentration of 1.8 μg/ml as a positive control to one well. After overnight incubation, the plates were developed and the number of spots was determined using the AID ELISpot Reader Unit (Autoimmun Diagnostika GmbH, Strassberg, Germany). Results were expressed as spot-forming cells per million input cells and were considered positive when exceeding all of the following criteria: (i) a minimum of five spots per well; (ii) a mean of negative wells plus three times the standard deviation of the negative wells; and (iii) three times the mean of the negative wells.
Kaposi's sarcoma-associated herpesvirus viral load determination
DNA was extracted from PBMC using the MagNAPure DNA kit (Roche Diagnostics, Basel, Switzerland). HHV-8 polymerase chain reaction used Taqman reagents with each reaction mixture containing extracted DNA from approximately 150 000 PBMC, 600 nmol each primer, and 200 nmol probe for the viral ORF25 as an internal standard . All reactions were performed in duplicate.
Cross-sectional differences in CD4 cell counts and viral loads between baseline, month 5 and month 11 were analysed using a Mann–Whitney test. The outcome of the two treatment groups was compared with a Fisher's exact test. For the longitudinal analyses, differences between baseline and month 11 for CD4 cell counts, HIV and KSHV viral loads and the immunological data were compared using a Wilcoxon signed-rank test for matched pairs. Correlations between viral loads, CD4 cell counts and virus-specific immune responses were tested using a Spearman's rank test.
Recovery of CD4 cells and suppression of HIV replication but not overall Kaposi's sarcoma-associated herpesvirus viremia in response to antiviral treatment
Thirty-three HAART-naive, HIV clade C-infected subjects presenting with KS were enrolled prospectively in Durban, South Africa, and were randomly assigned into two study arms, receiving either HAART alone or HAART in combination with chemotherapy. The clinical outcome was assessed after 11 months of treatment and showed overall significant reductions in HIV viral loads and increases in CD4 cell counts (P < 0.001, Table 1 and Fig. 1). KSHV viral loads remained high between baseline (median 758 copies/106 PBMC, n = 21) and 5 months (1199 copies/106 PBMC, n = 14) and then dropped to 56 copies/106 PBMC at 11 months (n = 17, Fig. 1). Although almost twice as many subjects showed undetectable KSHV viral loads at 11 months compared with baseline or 5 months, the drop in KSHV viral load did not reach statistical significance in this cross-sectional analysis.
Clinical outcome associated with CD4 cell count but not Kaposi's sarcoma-associated herpesvirus viral load at baseline
The clinical outcomes of the enrolled KS patients were compared with baseline CD4 cell counts, KSHV viral loads and virus-specific immune responses. In line with previously reported data, the baseline CD4 cell count was a determinant for disease outcome, as subjects with unfavorable or fatal disease progression had a significantly lower CD4 cell count at baseline (median 35, range 1–1080) than complete responders (median 227, range 106–501) or partial responders (median 135, range 4–618), P = 0.03 and P = 0.04, respectively . The baseline KSHV viral load did not differ significantly between the three groups although the complete responders had a higher median viral load than partial or non-responders (median KSHV viremia in complete responders 3485, partial responders 897 and progressors 332 copies/106 PBMC, respectively).
When the 33 subjects were stratified on the basis of the two different treatment regimens and analysed for disease outcome at 11 months, a trend towards a better disease outcome was observed with combination therapy, as this led more frequently to complete clinical responses (44%) and less frequent progressive disease (12%) compared with HAART-only treatment (17 and 24%, respectively, Fig. 2). Of note was the fact that regardless of whether or not patients were treated with combination therapy or HAART alone, 82% of all study subjects (n = 27) had an advantageous outcome (complete or partial response). These HIV clade C and non-Caucasian-based results are in line with data from studies performed in clade B infection and in regions of only low KSHV seroprevalence in the general population .
HAART treatment associated with significant increase in Kaposi's sarcoma-associated herpesvirus-specific cellular immunity over time
To investigate whether increased CD4 cell counts and relative KS disease control upon HAART initiation were associated with the reconstitution of specific antiviral immunity, KSHV, EBV and HIV-specific cellular immune responses were assessed. Peptide sets including 309 overlapping peptides spanning three lytic (K8.1, ORF57, ORF65) and three latent KSHV proteins (K10.5, K12, ORF73), as well as 220 HIV and EBV-derived peptides were used in ex-vivo IFN-γ ELISpot assays . PBMC samples from baseline and 11-month follow-up visits were available for 24 subjects; an additional sample at 5 months was available for immune analysis in 18 of the 24 individuals. For both KSHV and EBV, a significant increase in the magnitude of virus-specific ex-vivo T-cell responses was observed between baseline and 11 months (KSHV P = 0.0068; EBV P = 0.015, Fig. 3). At the same time, the magnitude of the HIV-specific immune response did not differ significantly. While KSHV-specific immune reconstitution was also reflected in an overall direct correlation between CD4 cell counts and KSHV-specific immune responses (P = 0.048, Fig. 3c), there was no significant association between KSHV viral loads and the magnitude of the virus-specific cellular immunity. A protein-specific breakdown of the KSHV responses indicated that both lytic and latent antigen-specific responses contributed similarly to the overall increased responses between baseline and 11 months (P = 0.062 for lytic and P = 0.048 for latent, Fig. 3d). To assess the kinetics of this virus-specific immune reconstitution, responses were assessed in a subset of 18 of the 24 individuals for whom an additional 5 months timepoint sample was available. Despite the smaller number of individuals included, a significantly increased magnitude of the KSHV-specific cellular immunity was detected between baseline and 11-month samples but not between baseline and 5 months, in line with the previously recorded ‘late’ reconstitution of the KSHV-specific immune response in HAART-treated KS patients (Fig. 3b) . No statistically significant difference in the magnitude of the KSHV-specific immune response based on disease outcome was observed in our cohort, however, probably also as a result of the small number of individuals with progressive disease for whom 11-month samples were available.
HAART combined with chemotherapy reduces Kaposi's sarcoma-associated herpesvirus viremia more efficiently than HAART alone
Whereas immune data were obtained at baseline and 11 months for 24 subjects, KSHV cellular viral loads for both these timepoints were only available for 14 individuals. Of these, each half was treated with HAART alone (n = 7) or with HAART plus chemotherapy (n = 7). The magnitude of the KSHV-specific immune responses at baseline and 11 months in these two subgroups did not show any statistically significant differences. Despite the small number of individuals included, subjects treated with combination therapy but not those with HAART alone showed a significantly reduced KSHV load over the 11-month follow-up (Fig. 4).
Although anti-HIV treatment has been shown to lead to a regression of overt KS, the factors controlling KSHV in HIV-infected individuals are poorly understood. Aside from general immune reconstitution that may restore the host's ability to control viral co-pathogens, some treatment strategies may also act directly on the co-pathogen replication, thus synergistically contribute to the control of KS. Of the individuals included in the present study, 78% showed undetectable HIV viremia and a significant increase in CD4 cell counts over the first 5 months of therapy with HAART with or without chemotherapy. The significant virological and immunological benefits were temporally associated with successful KS management in 82% of the enrolled subjects after one year, independently of chemotherapeutic treatment. Of note is the fact that in the present study half of the subjects were treated with HAART combined with a classic regimen of ABV-based chemotherapy. Although it is of low cost, conventional ABV treatment is currently limited to cases of more advanced or widespread KS disease [3,28], but because of its notable and often intolerable toxicity, the ABV combination regimen has been largely replaced by the more expensive pegylated-liposomal doxorubicin monotherapy or other single-agent regimens with paclitaxel . Nevertheless, the present data indicate that this low-cost therapy is an effective option, which in combination with HAART may be used especially in resource-limited settings.
Despite its potential for the design of immune-based therapeutic interventions, the role of host virus-specific immunity in the control of AIDS KS has not been defined. In the past, the gradual recovery of KSHV-specific immunity upon HAART initiation has been addressed in analyses limited to small numbers of individuals only and focusing on single human leukocyte antigen class I-restricted, KSHV-derived cytotoxic T-lymphocyte epitopes . In addition, past studies have focused on HIV clade B-infected individuals, with presumably frequent Caucasian host genetics, and did not include immune analyses of other viral co-pathogens, not associated with the clinical condition for which treatment was initiated. The present work thus expands these studies considerably by including more than 500 peptides that, besides EBV and HIV viral proteins, cover three lytic and latent KSHV antigens each, and by testing samples from 33 subjects followed longitudinally. The findings are in line with the report by Bourboulia et al. , showing a late recovery of KSHV-specific immunity in subjects undergoing HAART treatment. Our data also show that detectable KSHV-specific T-cell responses are becoming increasingly stronger after 5 months of treatment, in parallel with recuperating cellular immunity to EBV-derived antigens. The relatively weak responses seen to EBV may be explained by the use of pre-defined, EBV-derived cytotoxic T-lymphocyte epitopes that have largely been defined in the context of Caucasian human leukocyte antigen class I alleles and which may thus be less frequently targeted in this exclusively Zulu/Xhosa-based cohort . In contrast to EBV and KSHV, HIV-specific responses did not increase significantly over time, probably reflecting a balance between virus-specific immune reconstitution and the simultaneous suppression of viral antigen availability by HAART [29–31]. Together, it is unclear how general immune reconstitution contributes to the overall increased T-cell response, and to what degree the magnitude of these responses is driven by viral loads and antigen availability . Clearly, detailed analyses that separate the direct effects of HAART on CD4 cell counts from immune-mediated control of viral replication will be needed to address these questions.
The present data also show a pronounced decline in KSHV viremia in patients treated with combination therapy of HAART and chemotherapy. As the viral loads, but not the cellular immune responses, differed significantly at the 11-month timepoints between the two treatment groups, the data suggest that that antineoplastic agents could have direct effects on KSHV replication. It has been demonstrated that liposomal doxorubicin can decrease KSHV viremia , and that drugs such as methotrexate or protease inhibitors can exert anti-angiogenic effects, directly affecting viral replication [34–36]. Moreover, KSHV viral loads have been found to correlate with the extent of KS lesions [37,38], suggesting that the chemotherapeutic compounds in the ABV regimen could exert a direct antiproliferative effect on transformed KSHV-infected cells, thereby limiting viral replication. Therefore, together with general immune reconstitution under HAART treatment, the combined HAART and chemotherapy treatment might provide lasting immune control of KSHV, which a longer follow-up of the present cohort may allow us to determine.
Sponsorship: This study was supported by the South African National Research Foundation (Thuthuka grant GUN 2054349) and an ACRiA grant to A.M. F.B. was supported by a grant from the Swiss National Science Foundation (SNF-PBSKB-102686).
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Chemotherapy; cytotoxic T lymphocytes; Kaposi's sarcoma; Kaposi's sarcoma-associated herpesvirus; viral immune control; viral load
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