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Partners AIDS Research Center and Endocrine Unit, Massachusetts General Hospital, Charlestown
Howard Hughes Medical Institute, Boston
Department of Epidemiology and Biostatistics, University of CaliforniaSan Francisco
Blood Systems Research Institute, San Francisco
Departments of Microbiology and Medicine, Myles H. Thaler Center for AIDS Research, University of Virginia Health Systems, Charlottesville
Centers for Disease Control and Prevention, Atlanta, Georgia
AIDS Malignancy Consortium, University of Alabama, Birmingham
Cellular immune responses to Kaposi sarcomaassociated herpesvirus (KSHV), the etiological agent of KS and several other malignancies, are incompletely characterized. We assessed KSHV-specific interferon- enzyme-linked immunospot responses in a cohort of 154 individuals, using overlapping peptide sets spanning the KSHV-encoded latency-associated nuclear antigen (ORF73) and the minor capsid glycoprotein (ORF65). Among KSHV-seropositive subjects, ORF73-specific responses dominated over responses to ORF65 and were preferentially detected in human immunodeficiency viruscoinfected individuals who had elevated levels of cell-associated KSHV DNA, indicating that the viral antigen burden may have been driving these responses. Responses to both ORF73 and ORF65 were also detected in several KSHV-seronegative subjects who were at increased risk for KSHV infection, which demonstrates that cellular immunity can be found in the absence of detectable humoral responses. These data have implications for the reliable identification of KSHV infection and may help guide the design of immune-based therapeutic and prophylactic interventions.
Kaposi sarcomaassociated herpesvirus (KSHV) is the etiological agent of several human malignancies, including KS, multicentric Castleman disease, and primary effusion lymphoma [1]. The increased occurrence of these malignancies in immunocompromised individuals suggests that viral immune control may be essential in preventing KSHV-associated diseases. Indeed, immune reconstitution has been linked to the remission of KSHV-associated malignancies, which provides further support for this argument [2]. However, few data exist on the kinetics, specificity, and dynamics of KSHV-specific cellular immunity, despite the potential usefulness of such information for the design of immune-based therapies for KSHV-associated diseases.
In individuals infected with Epstein-Barr virus (EBV) or HIV, an association between viral load and the magnitude of the virus-specific T cell response has been suggested [35]. However, the data are often conflictingsome studies have described a direct association between cytotoxic T lymphocyte (CTL) responses and viral loads, whereas others find no, or even an indirect, association [3, 4, 6, 7]. Also, the initiation of highly active antiretroviral therapy (HAART) has been associated with weaker HIV-specific CTL responses over time due to reduced antigen availability [8]. A recent report by Bourboulia et al. [9] addressed how the initiation of HAART could also affect the KSHV-specific immune response in subjects coinfected with KSHV and HIV. These data show that, despite decreasing the KSHV burden in both peripheral blood mononuclear cells (PBMCs) and plasma, the magnitude of the KSHV-specific responses, at least against some antigens, increased gradually. At the same time, the magnitude of the HIV-specific response remained stable, despite a successful reduction in HIV load [8, 9]. Thus, it is unclear whether high KSHV antigen loads are driving stronger T cell responses or whether strong cellular responses are associated with reduced viral loads.
Ideally, immune assays should include all viral antigens if they are to provide a full appreciation of the total virus-specific immune response. However, a common obstacle for immunological studies of herpesviruses is the size of their genomes, which precludes the comprehensive antigenicity analyses that are feasible for smaller viruses [6, 10, 11]. Despite this hurdle, CTL responses to KSHV lytic and latent proteins have been described [9, 1220], but little is known about their kinetics during infection, their relative immunodominance, or their association with KSHV loads. Furthermore, no study has addressed the issue of potential immune responses in subjects who are at increased risk for KSHV infection yet appear to be seronegative [21, 22].
The present study was designed to provide insight into the immunodominance of 2 KSHV proteins that generally induce detectable humoral responses: latency-associated nuclear antigen (open-reading frame 73 or LANA) and lytic minor capsid glycoprotein (ORF65 or MCP) [13, 23, 24]. Factors, including copy number of viral DNA and KS disease status, that could affect the frequency of detection of these T cell responses were assessed. Finally, KSHV-seronegative individuals at increased risk for KSHV infection were included in these analyses for the identification of their cellular responses [25, 26]. Our data show that HIV coinfection and active KS are factors that influence the rate of detection of T cell responses to ORF73 and ORF65. However, the data also show that cellular immune responses can be found in KSHV-seronegative individuals who are at increased risk for KSHV infection, which suggests that currently available serologic assays may be underestimating the true KSHV seroprevalence.
SUBJECTS, MATERIALS, AND METHODS
Study subjects.
Peripheral blood was obtained from a total of 154 subjects who were recruited from cohorts established through the AIDS Malignancy Consortium (AMC) or who were recruited at medical centers in the Boston or the San Francisco areas. The AMC cohorts included individuals enrolled in the AMC protocol 010 (cyclophosphamide, doxorubicin, vincristine, and prednisone and CHOP/rituximab treatment of HIV-associated non-Hodgkin lymphoma) and protocol 013 (treatment of patients with mucocutaneous AIDS-associated KS). All subjects provided informed consent for these studies.
KSHV serologic testing.
Infection with KSHV was determined by either the clinical diagnosis of KS or the serologic detection of antibodies to KSHV proteins. Coded, single-blinded serum samples were tested at the Centers for Disease Control and Prevention by 3 serologic assays: an ORFK8.1 ELISA, an ORF65 ELISA, and a whole-cell immunofluorescence assay (IFA), in which KSHV is induced to undergo lytic replication [21, 22, 27]. Specimens that were reactive in 2 of 3 assays or in the IFA alone at a dilution of 1 : 80 were considered to be seropositive. Subjects with a single positive response in only the ORFK8.1 or the ORF65 ELISA were categorized as indeterminate.
Virus quantification.
PBMC-associated KSHV DNA levels were determined by real-time polymerase chain reaction (PCR) with primers KS1 (5-AGCCGAAAGGATTCCACCAT-3) and KS2 (5-TCCGTGTTGTCTACGTCCAG-3), which yielded a 233-bp product of ORF26, as described elsewhere [28]. Results were normalized to account for the total input cellular DNA in the PCR, and the lower limit of detection was 4 viral copies/106 PBMCs [13]. HIV load was measured in plasma by use of a Roche-Amplicor assay with a lower limit of detection of 50 viral copies/mL.
Enzyme-linked immunospot (ELISPOT) assays.
Peptides spanning the entire ORF73 (1162 aa, 85 peptides) and ORF65 (227 aa, 21 peptides) amino acid sequences were synthesized as 22-mer peptides, overlapping by 12 aa. Peptide sequences were based on the KSHV sequence from the KSHV-infected BC-1 cell line reported elsewhere [10]. An additional 57-aa sequence (five 22-mer overlapping peptides) was synthesized for the ORF65 C-terminal end, to account for an ORF65 sequence variation in patients with multiple myeloma that has been described elsewhere [13]. Pools that contained 510 ORF73- or ORF65-derived peptides were tested by ELISPOT assay, as described elsewhere [6]. Results were expressed as the number of spot-forming cells per 106 input cells, with a cutoff for positive responses of a minimum of 5 sfu/well or responses exceeding the mean of negative wells plus 3 SDs, whichever gave the higher value.
ORF26 nested PCR.
DNA was isolated from 5 × 106 PBMCs by use of the Qiagen DNEasy Tissue Kit, per the manufacturer's instructions. The primers used for ORF26 were as follows: 5-AGCCGAAAGGATTCCACCAT-3 (forward) and 5-TCCGTGTTGTCTACGTCCAG-3 (reverse) for the first stage and 5-CTCGAATCCAACGGATTTGA-3 (forward) and 5-ATATGTGCGCCCCATAAATG-3 (reverse) for the second stage. Then, 100 ng of DNA was used for each first-stage reaction, and a 1 : 10 volume of each first-stage reaction was used as the template for the second-stage reaction. The cycling parameters were the same for both reactions: 94°C for 2 min; 30 cycles of 94°C for 10 s, 58°C for 15 s, and 72°C for 30 s; and 72°C for 5 min. All samples were also amplified with GAPDH primer sets, to control for the quality of the test DNA.
Statistical analysis.
Statistical analysis was performed by use of GraphPad Prism for Macintosh (version 3.0; GraphPad). Results are presented as median values, unless otherwise indicated. Statistical analyses included the nonparametric Spearman rank test (2-tailed) for correlations and Fisher's exact test for the analysis of responses between the different groups.
RESULTS
KSHV serologic results in subjects with KS and those with and without risk factors for KSHV infection.
A total of 154 subjects were recruited into the study and were grouped into 1 of 3 groups on the basis of risk factors associated with KSHV infection [24, 25]. The first group (n = 37) consisted of individuals with a history of KS and who were therefore known to be KSHV positive. The second group (n = 83) consisted of individuals at increased risk for KSHV infection, including men who have sex with men (MSM) and HIV-coinfected individuals. Subjects in the third group (n = 34) were considered to be low-risk subjects and did not include MSM or HIV-coinfected individuals (table 1). Plasma samples from all 154 individuals were subjected to serologic testing by use of the IFA and ORFK8.1 and ORF65 ELISAs. All but 1 of the 37 individuals with KS (97%) tested positive for KSHV antibodies, which is in agreement with the near-complete rate of detection of anti-KSHV antibodies in patients with KS [21, 22]. In the at-risk group, 45 subjects (54%) tested positive, 5 showed indeterminate serologic results, and 34 tested negative for KSHV-specific antibody responses. Of these 45 KSHV-seropositive subjects, 11 (24%) were HIV negative. Finally, 0 of 33 low-risk subjects tested seropositive.
Detection of KSHV ORF65 and ORF73 T cell responses in KSHV-seropositive subjects.
Although samples from all 154 subjects were tested by ELISPOT assay, initial immune studies focused on 82 subjects who were presumably KSHV infected, including those with a history of KS (n = 37) and those at risk who tested seropositive (n = 45). PBMCs from these KSHV-seropositive subjects were tested for responses against synthetic peptides spanning the entire length of KSHV ORF73 and/or ORF65 by an IFN- ELISPOT assay. Of the 82 subjects, sufficient cells were obtained from 45 to test for responses against both antigens. Of the remainder, 32 samples were tested for responses only against ORF73, and 5 samples were tested for responses only against ORF65. Overall, responses to at least 1 antigen were detected in 37 (45%) of 82 subjects; 34 (44%) of 77 subjects tested for responses against ORF73 had detectable responses, and 12 (24%) of 50 subjects tested for responses against ORF65 had detectable responses (figure 1). Of the samples from 45 subjects that were tested for both antigens, 22 (49%) responded to ORF73, whereas only 12 (27%) responded to ORF65. Because 9 of 12 subjects who had responses to ORF65 also had responses against ORF73, responses to ORF65 were a good predictor for having responses against ORF73, which supports the immunodominant role for ORF73, relative to ORF65 (P = .026).
However, the immunodominance of ORF73 over ORF65 was less pronounced when subjects were analyzed separately, depending on whether they presented with KS. Of the subjects who presented with active KS, responses against ORF73 were detected in 59% of those tested, whereas 42% of the KS-free subjects mounted ORF73-specific T cell responses. Although this was not significantly different, significantly more individuals with active KS (33%) had detectable responses to ORF65 than did subjects without active KS (12%; P = .035). The data show that subjects with active KS had more readily detectable responses than did those who did not present with active KS, mainly because of an increased rate of response against the lytic-expressed ORF65 antigen. Interestingly, responses to KSHV-derived peptides were detected only in individuals coinfected with HIV; none of the 11 KSHV-positive, HIV-negative individuals responded to any of the KSHV peptides (P < .001). However, all 11 individuals had detectable responses against EBV-derived peptides (data not shown), which ruled out a general functional impairment in the PBMCs of these subjects [13].
Impact of KSHV copy number on KSHV-specific T cell responses.
Given that the development of KS has been associated with increased viral loads and elevated KSHV antibody titers [29, 30], the more frequent detection of T cell responses in the subjects with KS could potentially be due to elevated KSHV loads. Similarly, increased KSHV antigen load in the HIV-infected, KSHV-seropositive subjects may drive more readily detectable responses in HIV-coinfected subjects, compared with HIV-negative subjects. To test both of these possibilities, KSHV DNA copy numbers were compared between HIV-infected and -uninfected individuals, including all 11 KSHV-seropositive, HIV-negative subjects and 42 KSHV-seropositive, HIV-positive subjects (26 of whom had active KS). Indeed, significantly more KSHV genome copies were detected in PBMCs from HIV-positive subjects (median, 24 viral copies/106 PBMCs) than PBMCs from HIV-negative subjects (median, <3 viral copies/106 PBMCs; P = .041), which suggests that elevated KSHV DNA copy numbers in the HIV-coinfected subjects may have contributed to the more frequent detection of KSHV-specific responses in these subjects. Additionally, when the viral DNA copy numbers were compared between ELISPOT responders and nonresponders, regardless of their KS status, a 10-fold difference in the median KSHV copy number between ELISPOT responders (32 viral copies/106 PBMCs) and nonresponders (<3 viral copies/106 PBMCs) was observed (P = .035). The fraction of subjects with a detectable KSHV genome copy number was also significantly higher among the ELISPOT responders (60%), compared with nonresponders (31%; P = .035), which again suggests that elevated viral loads may be responsible for the more readily detectable ELISPOT responses (data not shown). This was further supported by the findings in 3 individuals for whom KSHV copy number and ORF73- and ORF65-specific responses were assessed longitudinally. Results showed that changes in the magnitude of ORF73- or ORF65-specific T cell activity were paralleled by changes in the amount of cell-associated KSHV DNA in PBMCs (figure 2). In all 3 subjects, the amount of KSHV DNA varied substantially among time points, and, in all 3 instances, the strongest cellular immune responses were detected at time points when increased levels of KSHV DNA were detected. Importantly, the cellular response to selected EBV peptides remained stable over time, independent of changes in the amount of KSHV DNA (data not shown), which suggests that KSHV loads and not the general immune status were responsible for fluctuations in the KSHV-specific T cell responses.
ORF73- and ORF65-specific responses in KSHV-seronegative and -seroindeterminate subjects.
The present cohort included 67 KSHV-seronegative and 5 seroindeterminate subjects, who were all also tested for cellular immune responses against at least 1 of 2 KSHV antigens (table 1). Of the 67 seronegative subjects, 34 were at low risk for KSHV infection and 33 were HIV-infected and/or MSM. Of these 72 subjects, sufficient cells were obtained from 35 to test for responses against both antigens. Of the remainder, 29 samples were tested for responses only against ORF73, and 8 samples were tested for responses only against ORF65. Positive responses by ELISPOT assay for responses against ORF73 and/or ORF65 were detected in 11 of 72 subjects. All were subjects who had at least 1 risk factor for KSHV infection, including 6 HIV-infected subjects and 5 MSM, of whom 3 were also HIV positive (table 2). Furthermore, the 11 ELISPOT responders included 2 of 5 subjects with indeterminate KSHV serologic results. Overall, the selective detection of responses in subjects at risk for KSHV infection suggested that these subjects may indeed be infected with KSHV and that, despite the negative serologic results at the time point tested, they were able to mount detectable cellular responses against KSHV.
To better determine the true KSHV infection status of these seronegative subjects, we attempted to amplify by PCR KSHV DNA from the 5 KSHV-seroindeterminate subjects, 8 KSHV-seronegative subjects (of whom 6 were HIV positive), and 17 KSHV-seropositive control subjects (all of whom were HIV positive). The PCR amplification detected viral DNA in only 20% of these samples, of which 5 were from KSHV-seropositive control subjects. The 1 positive PCR from a KSHV-seronegative sample was derived from a woman with HIV coinfection and detectable ORF73- and ORF65-specific responses. Thus, although the PCR amplification appeared to be even less sensitive than serologic testing, we were able to identify 1 individual who had negative KSHV serologic results but was PCR positive and who showed detectable KSHV-specific T cell activity (table 2) [31]. Thus, the combined ELISPOT and PCR data, as well as epidemiological considerations, strongly suggest that the subjects with positive ELISPOT responses were indeed KSHV infected but did not show a detectable serologic response at the time when mounting detectable T cell responses.
DISCUSSION
Cellular immunity to KSHV is poorly understoodfew reports have addressed the fine specificity, the kinetics, and the magnitude of KSHV-specific T cell responses. The present study was designed to overcome some of these gaps in our knowledge by examining KSHV ORF73- and ORF65-specific T cell reactivity in both KSHV-seropositive and -seronegative subjects at risk for KSHV infection. Our data support the hypothesis that the KSHV burden is an important determinant driving the virus-specific response, especially in subjects coinfected with HIV. However, our data also identified a number of KSHV-seronegative subjects with known risk factors for KSHV infection who mounted detectable T cell responses, which indicates that KSHV-specific responses can be detected in subjects who test negative in multiple serologic assays.
Overall, the serologic analyses of individuals enrolled in this cohort are in agreement with previously published reports. The sensitivity of the assays used was highlighted by the 97% seroprevalence among the subjects who presented with active KS [21, 22]. Both the 54% seropositivity rate among the subjects in the at-risk group, many of whom were HIV coinfected, and the negative serologic data in the low-risk group are in the range of previously published data [24, 25]. Interestingly, ORF73- and ORF65-specific responses were more frequently detected in HIV-coinfected subjects than in those with KSHV infection only, which initially raised concerns that the detected responses might reflect HIV-specific, rather than KSHV-specific, immune activity. However, several lines of evidence argue against such a conclusion: (1) subjects who had detectable responses against KSHV expressed a variety of HLA class I alleles (data not shown), making cross-reactive responses against a few HIV-derived CTL epitopes unlikely; (2) longitudinal analyses of samples from selected subjects revealed a temporal association between the KSHV, but not the HIV, load and the magnitude of detected responses; and (3) responses were detected in 2 HIV-uninfected subjects who were at risk for KSHV infection. Collectively, the data suggest that the detected responses were indeed KSHV specific and, on the basis of cell separation experiments in 3 individuals, mediated by CD8 T cells (data not shown).
The more frequent detection of KSHV T cell responses in these HIV-coinfected subjects could potentially be due to the direct activation of KSHV by HIV [32, 33] or to indirect mechanisms including HIV-mediated increased B cell activation [34], which may boost KSHV-specific T cell activity. Alternatively, KSHV-infected, HIV-uninfected subjects who effectively control KSHV replication could generate cellular responses against other regions of the KSHV genome instead of ORF73 and ORF65, which remains to be assessed.
The present data also reveal an increased response rate in subjects who presented with active KS and in those with elevated KSHV DNA copy numbers. This questions the in vivo effectiveness of these responses in controlling KSHV replication. Such "inefficient" CTL responses have been detected in other viral infections, such as HIV, where individuals with rapid HIV disease progression can mount strong CTL responses [6, 3537]. Future studies are required to determine why responses that are expanded in individuals with an elevated viral burden do not effectively exert immune control and how protective the detectable responses in individuals without active KS are. Such studies will also need to consider whether KSHV-specific T cells can effectively recognize KSHV-infected cells in vivo, despite the immunomodulatory effects of some KSHV gene products [3841]. However, the clinical improvements observed in patients with KS after immune reconstitution suggest that KSHV-specific immunity may contribute to effective in vivo immune control of this virus.
In addition to and although fluctuations in the amount of KSHV but not HIV load paralleled changes in the magnitude of KSHV-specific cellular responses in 3 subjects tested here, it is important to note that, in the present study, the "KSHV load" was measured as genome copy numbers in PBMCs and, hence, was reflective of cell-associated and potentially latent virus, rather than infectious virions in the plasma [42]. A better understanding of the relationship between viral load and CTL responses will therefore depend on the precise evaluation of viral antigen expression profiles, especially in body compartments such as the oral cavity, which has been shown to frequently harbor free virus [42]. Similarly, more-reliable markers of KSHV infection and broader CTL analyses are needed to permit a more comprehensive understanding of the relationship between KSHV-specific CTL immunity and the control of KSHV-associated diseases.
An important finding of the present study is the detection of KSHV-specific T cell responses in some individuals with a negative or indeterminate KSHV serostatus at the time when CTL responses were detectable. Because all 11 KSHV-seronegative/indeterminate subjects with detectable cellular responses were known to be MSM and/or HIV infected, they are likely infected with KSHV [22]. The detection of viral DNA in 1 of these subjects by a PCR-based assay demonstrates the presence of the virus and highlights the need for improved assays to more reliably determine KSHV infection. The need for more consistent measures of KSHV infection is also highlighted by the previously reported frequencies of seroreversion [15, 27], which may have caused some individuals to appear, at least temporarily, to be seronegative, whereas CTL responses remained detectable [43]. Clearly, more-extensive studies with prolonged follow-up and more repetitive serologic analyses will be needed to resolve the current discordance between cellular and humoral immunity to this virus. The experience from HIV infection and the conflicting data on cellular responses in highly exposed, persistently HIV-seronegative individuals suggest that this will not be a simple undertaking [44, 45].
Acknowledgment
We thank Lani Montalvo for assistance with the measurement of cell-associated KSHV.
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