Kaposi sarcoma herpes virus (KSHV), also known as human herpes virus 8, is the causative agent for Kaposi sarcoma. Historically, Kaposi sarcoma has been the leading cancer among persons infected with HIV (PHIV) , but a sharp decrease in the incidence of Kaposi sarcoma followed the introduction of HAART [2–5]. HAART use is even sufficient to induce remission in PHIV with Kaposi sarcoma . These phenomena are likely due to reconstitution of the immune system with concomitant improvements in KSHV-specific cellular  and/or humoral immunity and the ability to control KSHV viral replication . In line with these observations, HAART use among PHIV with Kaposi sarcoma has been associated with decreased KSHV viremia [9,10] and increased response to lytic, but not to latent, KSHV antibodies . Increased antibody response following HAART initiation has also been reported among PHIV without Kaposi sarcoma [8,12]. However, these studies had less than 30 patients, did not include information on KSHV viremia and could not explore the relationship between KSHV antibody response in relation to markers of HIV-related immunodeficiency such as CD4+ cell counts and HIV viral load.
In the present study, we aimed to better characterize the effect of HAART on KSHV antibody response and viremia among a large sample of MSM who first initiated HAART within the framework of the Swiss HIV Cohort Study (SHCS).
Patients were participants in the SHCS, an ongoing study that has been enrolling PHIV since 1984 from six large university hospitals, affiliated regional hospitals and private practitioners in Switzerland (www.shcs.ch). Detailed information on disease, laboratory tests and HIV-related treatments were collected at enrolment and at each 6-month follow-up visit. The present study was restricted to MSM in order to increase the proportion of KSHV-positive individuals and included information recorded in the SHCS database up to July 2006. To allow for a 24-month follow-up period, inclusion was restricted to MSM who were enrolled in the SHCS prior to July 2004.
A total of 1624 MSM initiated HAART while under active follow-up in the SHCS. From this group, we identified all 281 MSM who were naive to any other antiretroviral therapy and had serum samples available within 1 month prior to and 21–27 months after HAART initiation. Exclusion criteria were prior use of any antiretroviral therapy (878 patients), death (23 patients, including six Kaposi sarcoma), loss to follow-up (34 patients, including two Kaposi sarcoma) and unavailability of serum samples (358 patients). Among the final group of 281 MSM, 22 had or developed Kaposi sarcoma. Kaposi sarcoma was diagnosed within 3 months of HAART initiation in all but two participants, one diagnosed 8 months before, and the other 8 months after HAART initiation.
Kaposi sarcoma herpes virus serology, viremia and quantification
Serum samples stored at −80°C were retrieved from the SHCS repositories. All tests were performed at the Institute for Medical Microbiology, University of Basel, Basel, Switzerland. The presence of latent and lytic KSHV antibodies was assessed using immunofluorescence assays, according to published methodology . Samples showing fluorescence at 1 in 64 (starting dilution) were considered seropositive to lytic or latent KSHV antibodies, as described previously , and were not diluted beyond 1 in 32 768 (10 dilutions). Serum samples were also used to detect KSHV viremia by real-time PCR, as described elsewhere .
The SHCS has been approved by the ethical committees of all the collaborating clinics, and the present analysis was additionally approved by the scientific board of the SHCS and the ethics committee of the International Agency for Research on Cancer. Written informed consent is obtained from all SHCS participants in accordance with the Declaration of Helsinki.
Seroprevalence of latent and lytic KSHV antibodies and of KSHV viremia at HAART initiation were calculated for strata of Kaposi sarcoma, KSHV viremia, age at HAART initiation, HIV viral load, CD4+ cell count, CD8+ cell count and CD4+/CD8+ ratio. Differences between strata were compared by Cochran–Armitage trend test if a variable had more than two categories, or by χ2-test if it was dichotomous. At 24-month follow-up, changes in seropositivity for lytic and latent KSHV antibodies were evaluated by McNemar's test. Changes in antibody titers, as well as determinants thereof, were investigated in two ways.
In a first analysis, all participants were classified into those for whom antibody titers stayed the same/decreased (including those who remained seronegative or seroreverted) or increased (including those who seroconverted). The crude odds ratio (OR) comparing participants with increased titers to those with the same/decreased titers and the corresponding 95% confidence intervals (CIs) were calculated across strata of baseline characteristics.
In the second analysis, only participants with positive antibody results at both time points were included. Geometric mean titers (GMTs; log2) of latent and lytic KSHV antibodies at HAART initiation and at 24-month follow-up were compared using the nonparametric Wilcoxon signed-rank sum test, both overall and among participants with and without Kaposi sarcoma by CD4+ cell count (<50, 50–199, ≥200 cells/μl).
Mean values of CD4+ cell count, CD8+ cell count, CD4+/CD8+ ratio and HIV viral load were compared at HAART initiation and 24-month follow-up using the Wilcoxon signed-rank sum test. For each time point, these variables were also compared between participants with and without Kaposi sarcoma by the Wilcoxon–Mann–Whitney test. All P values are two-sided.
Of the 281 eligible MSM, nine with missing results for lytic and/or latent KSHV antibodies were excluded, leaving 272 MSM in the following analyses, including 22 with Kaposi sarcoma (20 stage T0 and two stage T1). Two Kaposi sarcoma patients received supplemental chemotherapy in addition to HAART, 18 were managed by HAART alone and no information was available for two patients. The mean age of the participants with and without Kaposi sarcoma at HAART initiation was 41 years (range 29–57) and 40 years (range 19–73), respectively. The mean CD4+ cell count among those with Kaposi sarcoma was significantly lower than in those without Kaposi sarcoma (100 versus 254 cells/μl; P = 0.0002), as was the CD4+/CD8+ ratio (0.13 versus 0.33; P = 0.0003). CD8+ cell counts and HIV viral loads were not different between the two groups (CD8+: 808 versus 872 cells/μl, P = 0.6; HIV viral load: 350 432 versus 396 512 copies/ml, P = 0.4).
At 24-month follow-up, participants showed substantial increases in mean CD4+ cell counts (from 242 to 501 cells/μl, P < 0.0001), CD8+ cell counts (from 866 to 1042 cells/μl, P < 0.0001) and CD4+/CD8+ ratios (from 0.3 to 0.6, P < 0.0001). HIV viral load decreased from 392 888 to 20 313 (P < 0.0001).
Kaposi sarcoma herpes virus antibody seropositivity and viremia at HAART initiation
At HAART initiation, 188 (69.1%) participants were seropositive to latent and 204 (75.0%) to lytic KSHV antibodies (79.4% were seropositive to either marker). Seropositive results for these two tests were correlated (Pearson r = 0.5, P < 0.001).
Table 1 shows the seroprevalence for each of the two antibodies and viremia at HAART initiation, stratified by age and other selected characteristics. Among participants without Kaposi sarcoma, 167 (66.8%) had a positive latent antibody response compared to 21 of 22 (95.5%) participants with Kaposi sarcoma (P = 0.005). For the lytic antibody, 182 (72.8%) participants without Kaposi sarcoma were seropositive, whereas all those with Kaposi sarcoma were seropositive (P = 0.005). Older age was significantly associated with seropositivity to lytic (P = 0.005), but not latent (P = 0.2) antibodies. High CD8+ cell counts were strongly associated with seropositivity to both latent (P = 0.001) and lytic (P = 0.006) antibodies, as were low CD4+/CD8+ ratios (P = 0.02 and 0.0004, respectively). There were no significant associations between either CD4+ cell count or HIV viral load and seropositivity to either KSHV antibody. These findings regarding KSHV seroprevalence were also observed when analyses were restricted to participants without Kaposi sarcoma (data not shown).
KSHV viremia was determined for 264 of the 272 participants, of whom 17 (6.4%) had detectable KSHV viremia (Table 1). All viremia participants were seropositive to lytic antibodies and 15 (88.2%) to latent antibodies. Presence of viremia increased with age (P = 0.01), was much higher in participants with Kaposi sarcoma than in those without (35 versus 4.1%; P < 0.0001), and was higher among those with HIV viral loads of 100 000 copies/ml or more (P = 0.0009). A higher prevalence of viremia at low CD4+/CD8+ ratios was of borderline significance (P = 0.06).
HAART-related changes in Kaposi sarcoma herpes virus antibody seropositivity and viremia
Following 24 months of HAART, seroprevalence of latent and lytic KSHV antibodies increased (Table 2). For latent antibodies, 22 participants seroconverted, whereas five participants seroreverted, so that seropositivity increased from 69.1 to 75.4% (P = 0.001). For lytic antibodies, 14 seroconverted and seven seroreverted, so seropositivity after HAART increased from 75.0 to 77.6% (P = 0.1). Seropositivity to both antibodies (from 64.7 to 70.2%, P = 0.001) and to either antibody (from 79.4 to 82.7%, P = 0.06) also increased (Table 2).
Of 17 participants with KSHV viremia at HAART initiation (mean viral load = 1738 copies/ml), only one remained viremia at 24-month follow-up (a Kaposi sarcoma case whose viral load increased from 704 to 7529 copies/ml). However, seven participants (none with Kaposi sarcoma) became newly viremia (Table 2), with a relatively low mean viral load of 462 copies/ml.
HAART-related changes in Kaposi sarcoma herpes virus antibody titers
Latent and lytic antibody titers both increased in 37.5% of participants (Table 3), although the changes in the two antibodies were not always consistent in the same participants. Compared with participants with neither Kaposi sarcoma nor KSHV viremia at HAART initiation, participants with Kaposi sarcoma showed an OR of 22.0 (95% CI 5.0–96.5) to have increased latent antibody titers and 5.1 (95% CI 1.9–13.6) to have increased lytic antibody titers. Those without Kaposi sarcoma who had KSHV viremia at HAART initiation were also more likely to have increased latent, but less so lytic, antibodies when compared with participants with no Kaposi sarcoma or KSHV viremia (Table 3). These results were robust after restriction to participants who were seropositive for both KSHV antibodies at both visits (n = 173; OR = 16.6; 95% CI 3.7–74.2 for participants with Kaposi sarcoma and OR 3.8; 95% CI 1.4–10.3 for participants with no Kaposi sarcoma but with KSHV viremia).
Participants with HIV viral loads of 100 000 copies/ml or more at HAART initiation showed an OR of 2.4 (95% CI 1.1–5.3) for an increase in lytic antibody titers compared to those with HIV viral loads less than 10 000 copies (OR = 3.4; 95% CI 1.2–9.3 among participants seropositive for both KSHV antibodies at both visits). The influence of HIV viral load on latent KSHV antibodies was similar to lytic antibodies, but did not reach statistical significance. There were no clear differences in the change in latent and lytic KSHV antibody titers by CD8+ cell count or by CD4+/CD8+ ratio (Table 3).
Titers for latent and lytic KSHV antibodies were more likely to increase among persons with CD4+ cell counts less than 50 cells/μl than in those with CD4+ cell counts of 200 cells/μl or more (Table 3). These results were similar when the analyses were restricted to participants without Kaposi sarcoma only (OR = 2.3; 95% CI 1.1–4.5 and OR 2.1; 95% CI 1.0–4.1, respectively) or to participants seropositive for both KSHV antibodies at both visits (OR = 3.1; 95% CI 1.3–7.1 and OR 2.5; 95% CI 1.1–5.8, respectively).
For 183 participants seropositive to latent antibodies both at HAART initiation and 24-month follow-up, GMTs increased from 402 (322–501) to 605 (478–766; P < 0.0001). For 197 participants seropositive to lytic antibodies at both points in time, GMTs increased from 331 (275–398) to 482 (396–588; P < 0.0001).
Figure 1 shows changes in GMTs between the two time points for four exclusive groups of participants: Kaposi sarcoma (irrespective of CD4+ cell count) and non-Kaposi sarcoma (stratified into three groups by CD4+ cell count). Among non-Kaposi sarcoma participants, low CD4+ cell counts predicted lower KSHV antibody titers at HAART initiation (Fig. 1). Increases in latent and lytic KSHV antibody titers were observed in all groups, but were greatest among participants with Kaposi sarcoma and among non-Kaposi sarcoma participants with CD4+ cell counts less than 50 cells/μl (Fig. 1). GMTs also tended to increase in all strata of CD8+ cell count and HIV viral load (data not shown).
To our knowledge, this study is the largest investigation into HAART-related changes in KSHV antibody response and viremia among MSM with and without Kaposi sarcoma. No clear evidence existed on whether the improvements in cellular immunity and control of viral infection induced by HAART would result in a decrease or increase in humoral immune response. Clearly, we observed increases for both lytic and latent KSHV antibodies, irrespective of how changes in antibody titers were evaluated.
The observed 69–75% seropositivity at baseline to one or both KSHV antibodies, even among participants without Kaposi sarcoma, suggests widespread exposure to this virus in Swiss MSM. The excess number of seroconversions over seroreversions following HAART suggests that some participants were already infected, but had undetectable titers at the time HAART was initiated (although we cannot rule out new primary infections). The seroprevalence of KSHV antibodies in the SHCS was thus at the upper end of previous estimates in HIV-infected MSM (range 29–62%) [14–27], and most closely resembles the findings from HIV-infected MSM in Italy (61–62%) [15,16], where KSHV antibody seroprevalence in the general population is known to be higher than in northern Europe and North America [28,29]. Differences in the sensitivity or specificity of the assays might have a role in these differences [30,31]. However, when the samples from participants without Kaposi sarcoma in this study were re-tested for specific antibodies against KSHV lytic antigen K8.1 , a similarly high seroprevalence of 79.6% was observed (data not shown). Among 34 HIV-infected MSM without Kaposi sarcoma in a previous study in the United States , KSHV viremia was detected in 5.9% of peripheral blood mononuclear cells (PBMCs), which is similar to the 4.1% in the present study.
In agreement with previous large studies of HIV-infected MSM [14,25], increasing age was a significant determinant of KSHV antibody seropositivity and KSHV viremia, but CD4+ cell counts were not. In addition, high CD8+ cell counts and low CD4+/CD8+ ratios were associated with increasing KSHV antibody seropositivity, and high HIV viral loads were correlated with the detection of KSHV viremia, which, to the best of our knowledge, has not been previously reported.
One of the most original findings of the present study was the HAART-related increases in KSHV antibody titers observed in a large group of participants without Kaposi sarcoma, for which there are few published data available for comparison. This phenomenon was reported for latent KSHV antibodies among eight patients in an earlier study . However, increases in KSHV antibody response were greatest among those participants who initiated HAART at lower CD4+ cell counts, irrespective of whether it was measured as the probability for antibody titers to increase (including seroconversion from previously undetectable levels), or as absolute increases in GMTs in consistently seropositive participants. Thus, although low CD4+ cell counts at HAART initiation predicted lower KSHV antibody titers among KSHV-seropositive participants, these initial differences were, to some extent, rescued at 24-month follow-up. In contrast, CD8+ cell counts were strongly correlated with KSHV antibody seropositivity at HAART initiation, but were not significantly associated with antibody titer increases.
The most important determinant of KSHV antibody seropositivity, antibody titers and viremia was confirmed to be the presence of Kaposi sarcoma [17,34,35]. Our study was not designed to focus on Kaposi sarcoma, but Kaposi sarcoma diagnosis was the trigger for HAART initiation in some participants. All Kaposi sarcoma cases were seropositive to at least one KSHV antibody, and one-third had KSHV viremia. These results are comparable to those seen in a recent study on Kaposi sarcoma prognosis in the SHCS, where 26% of 144 Kaposi sarcoma patients had KSHV viremia in serum at Kaposi sarcoma diagnosis and 82 and 93% were positive to latent and lytic KSHV antibodies, respectively . These results suggest that KSHV viremia might have some predictive value to identify underlying Kaposi sarcoma in HIV-infected MSM , in addition to its known predictive effect on Kaposi sarcoma prognosis [36,38,39].
Following HAART initiation in Kaposi sarcoma cases in the present study, increases in both latent and lytic KSHV antibody responses were striking, confirming the increases in latent antibody response seen in a previous study of 11 Kaposi sarcoma cases . These increases may contribute to Kaposi sarcoma remission, but they may also be a secondary effect of greater exposure to KSHV antibodies released by tumor lysis in the context of improving cellular immunity. In parallel, the detection of KSHV viremia declined to undetectable levels after HAART in all but one Kaposi sarcoma case, consistent with the findings of previous, smaller studies [8,10,12]. The use of sera rather than PBMCs may have reduced the sensitivity of viral DNA detection in the present study . Nevertheless, viremia was observed in 37% of Kaposi sarcoma cases, which is similar to studies using PBMCs among Kaposi sarcoma cases in Europe and the United States (range 29–37%) [33,37,40,41]. Moreover, it is known that viral DNA is not continuously detected in multiple samples taken from the same Kaposi sarcoma patient over time [38,40]. The figure of 37% is, however, very different from the 93% prevalence of KSHV viremia recently detected in PBMCs of Kaposi sarcoma cases in Uganda .
The present findings are likely to be relevant to HAART initiation among the millions of PHIV worldwide who live in KSHV endemic areas. In particular, exposure to KSHV in the general population of sub-Saharan Africa (30–80% KSHV antibody seroprevalence) [43–45] is as high as that seen in HIV-infected MSM in Switzerland, and Kaposi sarcoma has become the most frequent cancer in men and the third most frequent in women in the region .
In summary, we showed that after 24 months of HAART treatment, the humoral immune response to KSHV increases. This was observed both in a large group of MSM without Kaposi sarcoma and in Kaposi sarcoma cases in whom a parallel loss of detectable KSHV viremia was observed. Increases in humoral immune response may partly explain, together with changes in cellular immunity, the dramatic protection against Kaposi sarcoma offered by HAART, even when it is initiated at very low CD4+ cell counts . They may also apply to humoral responses to other viral infections among PHIV.
This study was funded by a grant from the OncoSuisse (ICP OCS 01355-03-2003).
This study was performed within the framework of the Swiss HIV Cohort Study, supported by the Swiss National Science Foundation. While at the International Agency for Research on Cancer, Robert Biggar was supported by an IARC International Expertise Transfer Fellowship and Sheena Sullivan was supported by a Raymond D. Goodman Scholarship (UCLA). The funders had no role in the design of the study; the collection, analysis and interpretation of the data; the decision to submit for publication; or the writing of the manuscript.
Author contributions: S.F. and G.M.C. conceived and designed the study. E.B.E.A., N.J.M., I.M. and M.R. acquired the clinical and epidemiological follow-up data. H.H.H and I.S. performed laboratory analyses. S.G.S., S.F., G.M.C. and R.J.B. analyzed and interpreted data. S.G.S. and G.M.C. drafted the article. All authors revised the manuscript critically for important intellectual content and approved the final version to be published.
The members of the Swiss HIV Cohort Study are M. Battegay, E. Bernasconi, J. Böni, HC Bucher, P. Bürgisser, A. Calmy, M. Cavassini, R. Dubs, M. Egger, L. Elzi, M. Fischer, M. Flepp, A. Fontana, P. Francioli (President of the SHCS), H. Furrer (Chairman of the Clinical and Laboratory Committee), C.A. Fux, M. Gorgievski, H.F. Günthard (Chairman of the Scientific Board), H.H. Hirsch, B. Hirschel, I. Hösli, C. Kahlert, L. Kaiser, U. Karrer, C. Kind, T. Klimkait, B. Ledergerber, G. Martinetti, B. Martinez de Tejada, N. Müller, D. Nadal, F. Paccaud, G. Pantaleo, A. Rauch, S. Regenass, M. Rickenbach (Head of Data Center), C. Rudin (Chairman of the Mother & Child Substudy), P. Schmid, D. Schultze, F. Schöni-Affolter, J. Schüpbach, R. Speck, P. Taffé, A. Telenti, A. Trkola, P. Vernazza, R. Weber, S. Yerly.
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