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 .
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.
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.
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