Short- and long-term effects of highly active antiretroviral therapy on Kaposi sarcoma-associated herpesvirus immune responses and viraemia
Bourboulia, Dimitraa; Aldam, Dianab; Lagos, Dimitriosa; Allen, Elizabethb; Williams, Ianb; Cornforth, Davidb; Copas, Andrewb; Boshoff, Chrisa
From the aWolfson Institute for Biomedical Research and the bDepartment of Sexually Transmitted Diseases, University College London, London, UK.
Requests for reprints to: C. Boshoff, Wolfson Institute for Biomedical Research, University College London, Gower St London WC1E 6BT, UK.
Received: 15 August 2003; revised: 9 September 2003; accepted: 15 October 2003.
Objective: To investigate the effect of highly active antiretroviral therapy (HAART) on Kaposi sarcoma-associated herpesvirus (KSHV) DNA load, anti-KSHV antibody responses and KSHV-specific CD8 T cell responses in HIV-infected individuals over a 2 year period.
Design: Prospective study of 27 HIV-infected antiretroviral therapy-naive individuals, with (n = 4) and without KS (n = 23), before HAART and at 3-month intervals, during treatment with HAART.
Methods: Sequential blood samples were collected for anti-KSHV antibody detection, KSHV DNA load in peripheral blood mononuclear cells (PBMC) and plasma, HIV Gag-specific and KSHV-specific CD8 T cell responses, HIV-1 plasma RNA load and CD4 and CD8 T cell counts.
Results: KSHV DNA in PBMC and plasma became less detectable over time during HAART, in particular after 12 months. KSHV DNA was undetectable in plasma after 24 months on HAART. Anti-KSHV lytic, but not latent, antibody levels increased within 12 months of treatment. KSHV-specific CD8 T cell responses were absent prior to HAART but became detectable in some patients within 6 months of starting treatment, and continued to increase thereafter.
Conclusions: HAART (both protease inhibitor-based and non-nucleoside reverse transcriptase inhibitor-based antiretroviral combinations) is associated with immune reconstitution to KSHV and with undetectable KSHV viraemia. However, this restoration is apparent (in particular) only after a relatively long (> 24 months) period of treatment. These immune responses could contribute to the decreased incidence of KS during HAART, but it is unlikely to be a complete explanation for the often rapid resolution of KS when HAART is started.
Kaposi sarcoma-associated herpesvirus (KSHV) is associated with all epidemiological forms of Kaposi sarcoma (KS) and with certain lymphoproliferations, such as primary effusion lymphoma and multicentric Castleman's disease [1,2]. KS is one of the most common tumours in Africa and the most common tumour found in patients with HIV-1 infection (AIDS-KS) . Prior to highly active antiretroviral therapy (HAART), detection of KSHV DNA in peripheral blood mononuclear cells (PBMC) from HIV-infected individuals predicted subsequent development of KS . Since 1996, HAART has been widely used and various hypotheses on how HAART acts in the resolution/prevention of AIDS-KS have been proposed [2,4,5].
We, and others, have previously demonstrated HLA class I-restricted KSHV-specific cellular immune responses against latent and lytic KSHV proteins in HIV-1-infected individuals with or without KS, and in HIV-uninfected adults with primary KSHV infection [6–9]. The present prospective study over 24 months analyses virological and immunological variables associated with KSHV immune restoration in 27 HIV-1-infected patients taking HAART. Anti-KSHV immune responses were assessed by measuring anti-latent and anti-lytic antibody levels, and CD8 T cell responses using a single-cell interferon γ (IFN-γ) enumeration assay. KSHV DNA load was measured in PBMC and plasma using real-time quantitative polymerase chain reaction (qPCR).
Patients and clinical samples
HIV-infected homosexual males attending a central London HIV outpatient clinic who, as part of routine clinical care, were to start HAART were invited to participate in the study. At study entry all were antiretroviral therapy naive and gave written informed consent. Twenty seven patients were recruited into the study; median age was 35 years (range, 27–55). Treatment response and safety of antiretroviral therapy were monitored as per routine clinical practice, with patients attending at baseline prior to starting HAART, at weeks 4 and 12, and every 3 months thereafter. The choice of HAART regimen was determined by the patient and supervising clinician in line with routine clinical practice guidelines. The median study follow-up period was 23 months (range, 0–38). Blood samples for the purpose of the study were drawn at baseline and every 3 months thereafter at the time of the routine clinic visit. Plasma and PBMC were isolated using Histopaque-1077 Hybri-Max gradient separation (Sigma-Aldrich, Poole, UK) for cryopreservation.
The study was approved by the Camden and Islington Community Health Service Local Research Ethics Committee and was conducted between 1999 and 2002.
HLA class I and II typing
DNA was extracted from 200 μl of plasma or cell pellets using the QIAamp blood Mini kit (Qiagen, Crawley, UK). All loci were simultaneously amplified in 144 sequence-specific primer reactions under a single set of conditions. The primers were designed using the amplification of refractory mutation system principle .
Interferon-γ release ELISpot assay and blocking experiments
Detection of single-cell IFN-γ release by ELISpot was performed as previously described . Briefly, 2 × 105 cells/well were cultured in 10% AB plasma/RPMI-1640 (Sigma) in a 96-well polyvinylidene difluoride-backed plate (Millipore, Bedford, Massachusetts, USA) coated with anti-IFN-γ monoclonal antibody (Mabtech, Nacka Strand, Sweden). Cells were stimulated with peptides (7 μmol/l) and phytohaemagglutinin (5 μg/ml; Sigma), in 10% AB plasma/RPMI-1640. Spot-forming cells (SFC) were detected according to the manufacturer's instructions (Mabtech). For each peptide, background values were subtracted and the number of spots was expressed as SFC/106 PBMC. Results were considered positive if the number of SFC/106 cells in peptide-stimulated wells was twofold higher than that in control wells (non-peptide-stimulated cells) and there were at least 50 spots.
Serology for KSHV
Antibodies to the latent nuclear antigen (LANA) were performed using a standard indirect immunofluorescence assay and antibody titres were measured . An adapted K8.1 ELISA was used for the detection of anti-KSHV lytic antibodies .
Quantification of HIV-1 RNA and blood cell counts
Plasma HIV-1 RNA loads were determined by the HIV-1 RNA 3.0 (bDNA) assay (Bayer, Newbury, UK). The lowest level of detection was 50 copies/ml.
Detection and quantification of KSHV DNA
A real-time quantitative PCR (TaqMan) assay was developed for the detection and quantification of KSHV based on a previously described method . Oligonucleotides for the LANA gene of KSHV and the human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene were designed using Primer Express (Applied Biosystems, Warrington, UK) software program. The forward and the reverse primers for LANA were 5′-TTGCCACCCACGCAGTCT-3′ and 5′-GGACGCATAGGTGTTGAAGAGTCT-3′; the TaqMan probe was 5′-FAM- TCTTCTCAAAGGCCA CCGCTTTCAAGTC-3′ TAMRA. The primers 5′-GGAGTCAACGGATTTGGTCGTA-3′ and 5′-GG CAACAATATCCACTTTACCAGAGT-3′ and the TaqMan probe 5′-FAM-CGCCTGGTCACCAGGG CTGC-3′ TAMRA were designed for the GAPDH gene. The mixture contained 10 μl DNA solution, 5 μl of 10× TaqMan buffer, 5 mmol/l MgCl2, 4 μl deoxynucleoside trisphosphate mixture (2.5 mmol/l dATP, 2.5 mmol/l dCTP, 2.5 mmol/l dGTP, 5 mmol/l dUTP), 700 nmol/l each primer, 200 nmol/l GAPDH and 150 nmol/l KSHV-LANA probes, 0.5 U AmpErase uracil N-glycosylase and 1.25 U AmpliTaq Gold (TaqMan PCR Core Reagent Kit, Applied Biosystems). Both amplifications were carried out under similar conditions . The amplification and analysis were carried out using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems).
The peptides were synthesized using fluorenylmethoxycarbony solid-phase chemistry. Identified A*02- restricted KSHV peptides derived from the following proteins: K12 (LLNGWRWRL) , gH (FLNWQ NLLNV) , gB (LMWYELSKI)  and K8.1 (gp 35/37) (LVLILYLCV), which is a hybrid 9-mer from two overlapping K8.1 15-mers WAVGLLLGLV LILYL and the originally identified A*02-restricted 15-mer LILYLCVPRCRRKKP . The HLA-A*0201-restricted HIV-1 peptide SLYNTVATL from p17 (SL9, amino acid residues 77–85) was used to determine the frequency of Gag-specific IFN-γ-secreting cells.
To provide summary statistics over the study, time was grouped as baseline (initial), 1–6, 7–12, 13–24, and > 24 months. In each time period, with the exception of baseline, patients may have contributed more than one measurement. The summary statistics were calculated from all available measurements. The missing data have been treated as missing completely at random, since in this study the number and timing of tests performed are considered unrelated to the values of the various measurements studied for each patient. At baseline, differences between patients with and without KS and differences between patients receiving non-nucleoside reverse transcriptase inhibitors (NNRTI) or protease inhibitors (PI) were tested by using the Mann–Whitney test. To test for and estimate the magnitude of change over time, binary, ordinal and continuous outcomes were analysed using logistic, proportional odds and linear regression, respectively. For each outcome, the difference between baseline and all follow-up times was tested, and a gradual trend over time was assessed. This provided odds ratios (OR; logistic and proportional odds regression) and mean outcome differences (linear regression) as the measures of effect. The generalized estimating equation methodology  was used to account for the correlation between measurements from the same patient, with the exception of the ordinal outcomes, where for computational simplicity the related survey methodology of STATA 7.0 (Stata Corp., College Station, Texas, USA) was used. An exchangeable working correlation was selected for the generalized estimating equation, and the robust variance estimator was used. All analyses were performed in STATA 7.0.
Patient characteristics at baseline
The baseline patient characteristics (prior to initiation of HAART) are shown in Tables 1 and 2. Four patients had cutaneous KS at study entry. The median CD4 T cell count was significantly lower in patients with (n = 4) than in those without (n = 23) KS (P = 0.03) (data not shown). Following baseline blood sampling, six patients received a PI and 21 a NNRTI, each in combination with two nucleoside analogue reverse transcriptase inhibitors as their HAART regimen (Table 1).
Antibody responses and KSHV DNA detection at baseline
Prior to starting HAART, 20/27 (74%) individuals had detectable LANA antibodies (Table 2) but only 12 (44%) had detectable anti-lytic (K8.1) antibodies. Three out of four (75%) patients with KS had antibodies to either LANA or K8.1 antigens (data not shown).
KSHV DNA load was determined in both PBMC and plasma (Table 2). Before HAART, 14/27 (52%) showed detectable KSHV DNA in PBMC, whereas only 7/27 (26%) had detectable KSHV DNA in plasma. Patients with KS had lower median numbers of KSHV copies in PBMC (17 copies/105 cells) and in plasma (948 copies/ml, one individual) compared with those without KS (57 copies/105 cells and 6686 copies/ml plasma, respectively), although the number of patients with KS (n = 4) is too small to suggest a significant finding.
Anti-KSHV antibody responses during therapy
The effects of HAART on anti-KSHV antibody responses were determined by studying changes in the percentage of positive samples and in the antibody levels (titres and absorbances) as a trend over the 24 months on HAART, and also by comparing all values after baseline with those at baseline (Table 3). Overall, no significant changes and specific trends were observed for the anti-LANA antibody responses after the initiation of HAART until the end of the study. However, the percentage of samples positive for anti-K8.1 antibodies increased somewhat with time (Table 3). These data suggest that the average levels of anti-K8.1 lytic antibodies may increase during the first 12 months of HAART, but they subsequently decline, causing an apparently negative trend over time.
Herpesvirus DNA detection during therapy
KSHV DNA load was measured in all PBMC and plasma samples provided after baseline (Table 4). When the effect of HAART at all time points after baseline versus the baseline was examined, there was a decrease of KSHV detectability in PBMC samples, but this was not significant [OR 0.62; 95% confidence interval (CI), 0.25–1.53]. However, when the gradual trend of KSHV detectability in PBMC was examined over the 1 year period, it was significantly less detectable (OR 0.40; 95% CI, 0.26–0.63) (Table 4).
KSHV DNA load in plasma was also analysed (Table 4). Both the percentage of plasma samples with detectable KSHV DNA and the amount of virus detected were significantly reduced during HAART. Within 12 months, only 12.5% of all samples had detectable KSHV viraemia compared with 25.9% at baseline, and this percentage decreased further (6%) during the second year. KSHV DNA became undetectable in plasma after 24 months on HAART. The amount of virus detected in plasma also significantly declined, both when considering all measurements after therapy versus baseline mean log10 KSHV DNA load, and over time (Table 4).
At baseline, seven patients had no detectable antibodies to KSHV latent or lytic antigens. However, five of these had detectable KSHV DNA in PBMC or plasma, and the other two seroconverted during the study.
KSHV-specific CD8 T cell responses during HAART
To investigate the presence and function of KSHV-specific CD8 T cells, four previously identified KSHV-specific CD8 HLA-A*02-restricted cytotoxic T-lymphocyte (CTL) epitopes (nine amino acid residues long), derived from one latent (K12) and three lytic (K8.1, gB and gH glycoproteins) antigens, were used to stimulate PBMC from nine patients with HLA-A*02 and five without A*02, at baseline and during HAART. To detect and assess the function of antigen-specific CD8 T cells, the IFN-γ release ELISpot assay was used. None of the non-A*02-carrying patients showed any responses when their PBMC were stimulated with either the K12 or the K8.1 epitopes at baseline and during HAART (data not shown).
To assess the effects of HAART on the KSHV-specific CD8 T cell responses from the nine patients with HLA-A*02, the trend of the responses over time was calculated from all available measurements (Fig. 1). There were significantly more responses to K12 and K8.1 peptides in these patients compared with those not carrying HLA-A*02 (P = 0.007 for K12 and P = 0.013 for K8.1 peptides, respectively). At baseline, none of the nine patients with HLA-A*02 demonstrated CD8 T cell responses (> 50 SFC/106 PBMC) to the KSHV epitopes. In contrast, KSHV-specific CD8 T cell responses were demonstrated after therapy started (Fig. 1). There was a significant positive trend in the percentage responses to the K12 epitope (OR, 6.94 for a 1 year time interval; 95% CI, 2.24–21.52). Examination of the data suggests that this increase is particularly evident during the second year on HAART (range, 50–1365 SFC/106 PBMC). Responses against the K8.1 epitope appeared early on HAART and were maintained thereafter (range, 53–353 SFC/106 PBMC). The increasing trend in response to KSHV K8.1 epitope (OR, 1.65 for a 1 year time interval; 95% CI, 0.94–2.92) was not significant. KSHV-specific CTL epitopes from gB and gH antigens were also tested in a smaller number of patients (two patients were tested for responses to gB and five for responses to gH), because of a limited number of viable PBMC. One individual responded to gB at week 16 (53 SFC/106 PBMC) and one to gH at week 31 (50SFC/106 PBMC) (data not shown).
To verify that the recovery of CD8 T cell responses against the KSHV epitopes K12 and K8.1 represents the effects of HAART in all nine patients studied (i.e., the analysis is not biased towards a few patients that may have provided most of the analysed samples), the number of responders to these epitopes was determined at every time point (data not shown). This analysis showed that the recovery of CD8 T cell responses against KSHV epitopes occurred with similar trends in most A*02-carrying patients tested, confirming the results in Fig. 1.
Responses to KSHV in patients with AIDS-Kaposi sarcoma
Four individuals who enrolled in the study had KS. Three patients had complete remission of KS lesions on HAART and one patient had stable disease. Two patients also received chemotherapy (Table 1). All four responded to HAART, showing HIV- and KSHV-specific immune reconstitution. This was mainly evidenced by the significant increase of KSHV-specific CD8 T cell responses during the first 6–9 months on HAART, which persisted (one patient did not have A*02 and was not included in the CD8 T cell response follow-up). Furthermore, KSHV DNA in plasma became undetectable in three out of four patients within 12 months; the fourth patient had undetectable virus in plasma after 23 months on HAART. Finally, KSHV DNA was detectable in PBMC of all four patients in most time points tested. There was not any significant trend in the anti-KSHV humoral responses and all four had detectable anti-LANA and/or K8.1 antibodies throughout the study. However, larger cohorts of HIV-infected individuals with KS need to be studied in order to draw conclusions about the long-term effects of HAART on AIDS-KS.
Responses to HIV infection at baseline and during therapy
Prior to HAART, the median plasma HIV RNA level in all 27 patients was 155 050 copies/ml (range, 4100–358 100) (Table 2). The median CD4 T cell count was 200 × 106 cells/l (range, 10–350) and the median CD8 T cell count was 925 × 106 cells/l (range, 330–2840). Excluding the four patients who provided study bloods at baseline only, plasma HIV RNA levels became undetectable (< 50 copies/ml) in all other patients taking HAART (22 within 6 months) and was paralleled by an increase in CD4 T cell counts. No individual experienced viral load rebound whilst on HAART during the study.
CD8 T cell responses to HIV were measured using an immunodominant HLA-A*02-restricted CTL epitope associated with chronic HIV infection. Significant responses to HIV Gag SL9 epitope were demonstrated even prior to initiation of HAART (median 350 SFC/106 PBMC; range, 60–763); 33% of patients tested showed responses to the epitope prior to HAART but no definite trend was demonstrated during HAART (OR, 1.23 for a 1 year time interval; 95% CI, 0.74–2.04) (Fig. 1).
Four patients provided only baseline samples. In case of any bias from their inclusion, the analysis was repeated excluding these four patients but with no appreciable differences in the findings.
The aim of this study was to investigate the effects of HAART on KSHV infection, in the presence of HIV-induced immunosuppression and in association with the functionality of the immune system.
Detection of anti-LANA antibodies is primarily used as a marker of an established persistent latent KSHV infection. LANA is one of the few viral proteins expressed during latency and one of the most immunogenic KSHV antigens. HAART had little effect on the detection or titre of the anti-latent KSHV LANA antibodies (Table 3). However, antibody levels against EBNA-1, a functional homologue of LANA, and EA (early antigen) of Epstein–Barr virus have been shown to increase significantly during HAART . In particular, EBNA-1 has been correlated with immune reconstitution and the control of Epstein–Barr virus replication, and decrease of anti-EBNA-1 IgG levels has been associated with increased immunosuppression .
In contrast, the anti-lytic KSHV K8.1 antibody responses increased during the first 12 months of HAART (Table 3). K8.1 is the most immunogenic envelope glycoprotein of KSHV . We have previously suggested that anti-lytic KSHV antibodies might be more effective in the control of KSHV pathogenesis than anti-LANA antibodies . Even though the percentage of samples with detectable antibodies against K8.1 increased somewhat during HAART, the average levels declined after the first 12 months on therapy. This may suggest a gradual reduction of antigenic stimulation owing to decreased KSHV lytic viral replication and, therefore, decreased number of virions in the circulation. The increase of anti-lytic antibody responses observed during the first 12 months on HAART (Table 3) concurs with the significant decrease in plasma KSHV DNA load over time (Table 4), and as such anti-KSHV lytic responses may be important in the control of KSHV lytic replication.
The percentage of PBMC samples at baseline with detectable virus was similar to previous reports, when real-time PCR was used as the detection method . The percentage of PBMC samples with detectable KSHV DNA decreased significantly during the study (Table 4). In plasma, the number of samples with detectable KSHV DNA and the level of viraemia decreased significantly over the period of the study. Eventually, KSHV DNA in plasma became undetectable after 24 months on HAART (Table 4). It is also of note that the majority of our patients were receiving NNRTI- and not PI-based triple therapy (Table 1). This confirms previous results  that PI are not essential to clear KSHV DNA load as some had suggested . In addition, we monitored eight individuals up to 38 months on HAART and at that stage KSHV DNA was only detected in one PBMC sample (68 copies/105 PBMC) (Table 4). This shows that HAART can suppress KSHV replication for a prolonged period of time, agreeing with the decreased incidence of KS development in patients taking HAART [23,24]. In our study, three patients had complete remission of KS lesions during HAART, whereas one had stable disease. In contrast, Epstein–Barr virus load in PBMC in HIV-infected individuals remains stable and can occasionally increase during HAART . This could partially explain why the incidence of HIV-associated non-Hodgkin's lymphoma declines to a lesser extent than KS [26,27]. Although it was proposed that HIV-1 Tat could play a role in KS development, it has as yet not been shown that reduction of HIV-1 Tat levels during HAART suppresses KSHV viraemia, resulting in the resolution of KS [4,28].
Finally, we studied KSHV CD8 T cell responses against previously defined HLA-A*02-specific CTL epitopes [9,15,16]. By measuring IFN-γ release, we were able to study whether the functionality of KSHV-specific CD8 T cells was improved with HAART. The ELISpot assay is reliably used in the identification and characterization of CD8 T cell responses to HIV antigens during acute and chronic infection , and to Epstein–Barr virus antigens [30,31]. ELISpot assays are used for the evaluation of HAART-induced immune restoration, vaccine design against HIV  and to Epstein–Barr virus antigens for the monitoring of adoptive cellular immunotherapy .
Prior to therapy, none of the nine individuals with HLA-A*02 studied showed significant CD8 T cell responses against the known KSHV peptides, whereas such responses increased significantly during HAART (Fig. 1). Some CD8 T cell responses to the lytic KSHV K8.1 epitope appeared within 6 months of starting HAART; their frequencies increasing further within 12 months (Fig. 1). Responses to the latent K12 epitope appeared to recover during the second year on HAART, with the frequencies significantly increasing during HAART (Fig. 1). Specific responses to gB and gH epitopes were almost absent during therapy (data not shown). However, studies on KSHV-seropositive non-HIV-infected individuals have shown significant responses to these epitopes [15,16]. Our data suggest the importance of the CD8 T cell responses against K12 and K8.1 for KSHV immune recovery. However, responses to one or two specific epitopes do not reflect the full repertoire of anti-KSHV immune responses. Therefore, future work has to determine whether other KSHV-specific epitopes are able to elicit potent CD8 T cell responses and contribute to the restoration of KSHV-specific T cells.
Although, HIV-specific HLA-A*02-SL9 CD8 T cell responses have been associated with the control of HIV replication during chronic HIV infection , three patients had significant Gag-specific CD8 T cell responses at the beginning of HAART (Fig. 1). Patients in whom Gag-specific CD8 T cell responses were detectable maintained longitudinally these responses and no HIV rebound was observed, confirming the inverse association between HIV RNA load and these responses .
In conclusion, HAART promotes long-term KSHV immune reconstitution in patients with and without KS. This was demonstrated by a significant decline of KSHV DNA load in PBMC and plasma, an initial increase of anti-lytic antibodies and a significant increase of anti-KSHV-specific CD8 T cell responses within 12 months of starting HAART. However, prolonged HAART (more than 12 months) is necessary for these anti-KSHV effects to be established and maintained.
Sponsorship: This work was funded by the Medical Research Council UK, Cancer Research UK and Elton John AIDS Foundation.
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