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IMMUNOBIOLOGY
From the Department of Infectious Diseases and
Microbiology and the Department of Biostatistics, Graduate School of
Public Health, and the Department of Pathology, School of Medicine,
University of Pittsburgh, Pittsburgh, Pennsylvania; Department of
Epidemiology, The Johns Hopkins University, Baltimore, Maryland;
Centers for Disease Control and Prevention, Atlanta, Georgia; and
School of Veterinary Medicine, Louisiana State University, Baton Rouge,
Louisiana.
Human herpesvirus 8 (HHV-8) is a recently discovered
gammaherpesvirus that is the etiologic agent of Kaposi sarcoma (KS). The natural history of primary HHV-8 infection, including clinical outcome and host immune responses that may be important in preventing disease related to HHV-8, has not been elucidated. The present study
characterized the clinical, immunologic, and virologic parameters of
primary HHV-8 infection in 5 cases detected during a 15-year longitudinal study of 108 human immunodeficiency virus type 1 seronegative men in the Multicenter AIDS Cohort Study. Primary HHV-8
infection was associated with mild, nonspecific signs and symptoms of
diarrhea, fatigue, localized rash, and lymphadenopathy. There were no
alterations in numbers of CD4+ or CD8+ T cells
or CD8+ T-cell interferon Human herpesvirus 8 (HHV-8, also called Kaposi
sarcoma-associated herpesvirus) is a recently discovered
gammaherpesvirus that is the etiologic agent of Kaposi sarcoma
(KS).1 There is currently little knowledge of the clinical
syndromes associated with primary HHV-8 infection or immune responses
to the virus. Primary infection of adults with Epstein-Barr virus
(EBV), the other human gammaherpesvirus, can commonly result in a
prolonged, although self-limiting, symptomatic mononucleosis illness
with long-term expansion of activated CD8+ T cells and
inhibition of T-cell reactivity to mitogens and viral antigens.2-8 Recently, primary infection with HHV-8 was
reported in one adult patient infected with human immunodeficiency
virus-1 (HIV-1), which was associated with fever, arthralgia, and
cervical lymphadenopathy.9 It is not clear, however,
whether these clinical signs and symptoms were related to the
underlying HIV-1 infection in this patient.
Virus-specific CD8+ T cells are likely to be central in
host control of primary HHV-8 infection. CD8+ T cells
specific for lytic cycle proteins of EBV, including structural, immediate-early (IE), and early proteins,10-14 appear
prominent in EBV mononucleosis. Thus, they may play a major role in
limiting the primary phase of EBV infection.3,11,15
Indeed, 0.5% to 6%, and as much as 44%, of circulating
CD8+ cells are specific for EBV lytic peptides by HLA
tetramer staining during infectious mononucleosis.16,17 It
has also been shown that IE proteins are frequent targets for
EBV-specific, CD8+ T cells during persistent
infection.14,18 Similarly, infection of mice with murine
herpesvirus 68 (MHV-68), another gammaherpesvirus, results in expansion
of CD8+ cytotoxic T-lymphocyte precursors (CTLp's)
specific for MHV-68 lytic proteins.19,20 These are
detectable in both lymph nodes and at the site of local infection after
primary infection in mice,20 and could be involved in
control of MHV-68 infection.21,22 In contrast, only 2 studies to date have been able to detect CD8+ T-cell
reactivity specific for HHV-8.23,24 Moreover, the levels of anti-HHV-8 CD8+ T-cell responses measured by bulk CTL
assays used in these cross-sectional studies were relatively low,
with narrow specificity for different HHV-8 proteins.
In this report, we conducted the first prospective study of
HIV-1-negative adults with documented seroconversion to HHV-8 infection to identify clinical syndromes, potential immunosuppressive states and immune responses, and virologic markers related to this
primary gammaherpesvirus infection. We used CTLp and single-cell interferon Patients
Viral serology
An enzyme immunoassay (rLAV EIA, Genetic Systems, Redmond, WA) and an immunoblot assay (Biorad Laboratories, Hercules, CA) were used for detection of antibodies to HIV-1 as per the manufacturer's instructions. The 5 immunology study subjects were seropositive for cytomegalovirus (CMV) Abs by passive latex agglutination (Becton Dickinson, Mountain View, CA). Polymerase chain reaction assay for HHV-8 DNA DNA isolated from PBMCs was analyzed by polymerase chain reaction (PCR) for the presence of HHV-8 DNA. PCR analyses were performed using primers that amplified a 233-bp fragment of the minor capsid gene encoded by open reading frame (ORF) 26.27,28 For nested PCR experiments, 5 µL of each PCR reaction was amplified with a primer set located internal to the ORF 26 primers. The nested primers were 5'-ATGGTCGTGCCGCAGCAACTGG-3' and 5'-CGCCCCATAAATGACACATTGG-3'. Cellular DNA was isolated using the genomic DNA isolation kit from Gentra Systems (Minneapolis, MN). PCR was performed on samples equivalent to the amount of DNA from 5 × 104 or 2.5 × 105 cells. Amplification was done for 5 minutes at 94°C for 35 cycles with a single cycle consisting of 1 minute at 94°C, 1 minute at 58°C, 1 minute at 72°C, and 7 minutes at 72°C. PCR products were separated on 2% agarose gels, transferred to nytran membranes, and hybridized with an internal oligonucleotide probe labeled with 32P. The sequence of oligonucleotide probe was TGCAGCAGCTGTTGGTGTACCACAT. PCR products that hybridized to the probe were detected by image analysis with an Imagequant PhosphorImager (Molecular Dynamics, Sunnyvale, CA).Construction of recombinant vaccinia viruses containing HHV-8 lytic cycle proteins The ORF for glycoprotein B (gB; ORF 8) was obtained as the plasmid gB-Blunt-pCR. The ORFs for glycoprotein H (gH; ORF 22), major capsid protein (MCP; ORF 25), a minor capsid protein (MiCP; ORF 26), and an IE protein (ORF 57) were amplified by PCR using the HHV-8 genome contained in the BCBL-1 cell line as the template. The primer sets used for PCR amplification were: (gH) 5'-CACCTAGAGGATCCGACATGCAGGGTC, 3'-TAAAAAATCTAAAGCTTTATTGACCG; (MCP) 5'-CTCGAGCGCTGGATCCATGGAGGCG, 3'-CACGATGAAAGCTTTCGAGCC; (MiCP) 5'-GGCTAGTCATATGGCACTCGAC, 3'-CACGATGAAAGCTTTCGAGCC; and (IE) 5'-CTCCCTGCGAATTCGCATGATAATTG, 3'-GTTACATGGAATTCACGGGAGACAC. The amplified ORFs were cloned into the vaccinia virus shuttle vector, pSC11, and recombinant vaccinia viruses constructed by transfection of cells infected with wild-type vaccinia virus (WR) as described previously.29,30 Each virus was plaque purified 3 times. Rabbit polyclonal Abs against MiCP and mouse polyclonal Abs against MCP were generated by immunization of animals with purified bacterial fusion proteins. Mouse polyclonal Abs against IE protein were generated by immunization of animals with a peptide corresponding to amino acids EYYRPGDVMGLLNVLV (single-letter amino acid codes). Rabbit antiserum against gB and gH was generated against recombinant proteins. Identification of the HHV-8 proteins expressed by the recombinant vaccinia viruses was determined by immunoblot analysis of infected cell lysates as described previously.29,30 Protein bands corresponding to the predicted sizes of the individual proteins were noted in vaccinia virus recombinant-infected cell lysates as 120 kd (gB), 74 kd (gH), 150 kd (MCP), 32 kd (MiCP), and 27 kd (IE).Limiting dilution and bulk CTL assays The CTLp frequencies were assessed using a modification of our limiting dilution assay for HIV-1.31 On day 0, autologous, EBV-transformed B-lymphocyte cell lines (B-LCL) were infected overnight with recombinant vaccinia viruses in RPMI medium (Life Technologies, Gaithersburg, MD) supplemented with 15% fetal calf serum (FCS), at 37°C in 5% CO2. On day 1, these cells (stimulators) were inactivated with psoralen (10 µg/mL, Sigma, St Louis, MO) and long-wave UV light (40 mW/cm2) for 30 minutes, then resuspended at the concentration of 5 × 104 cells/mL in stimulation medium (RPMI 1640 medium with 15% FCS plus human recombinant interleukin 2 [IL-2; 100 U/mL; Chiron, Emeryville, CA]). Allogeneic PBMCs (feeder cells) from normal donors were -irradiated
(4000 Rad) and resuspended at 1 × 106 cells/mL. Frozen
PBMCs (effectors) were thawed and resuspended in stimulation medium at
different concentrations (160 000, 120 000, 60 000, 30 000,
10 000, 5000, 2500, and 0 [control] cells/mL). The mean ± SE
viability of the thawed PBMCs was 92% ± 1% (n = 37).
Stimulators, feeders, and effectors were plated into round-bottom 96-well plates (Becton Dickinson) in 50 µL, 50 µL, and 100 µL, respectively. Twenty-four wells were used for each different
concentration. The wells without effectors were used as the negative
controls. The plates were incubated at 37°C in 5% CO2
for 14 days, and the medium was changed every 3 to 4 days. On day 13, the targets were prepared by radiolabeling infected autologous B-LCL
with 51Cr (100 µCi/mL, Dupont NEN, Boston, MA) for 16 hours at 37°C in 5% CO2. On day 14, target cells were
washed and added to the effectors. The effector cells were divided into
2 sets, with one set added to 24 replicate wells containing VSC11
infected target cells as controls for 4 hours at 37°C in 5%
CO2, and the other set incubated under the same conditions
with targets cells infected with vaccinia virus vectors expressing
individual recombinant HHV-8 proteins. The plates were then harvested
and counted for radioactivity in a gamma counter. Mean spontaneous
release and mean maximum lysis were calculated for wells with targets
alone and with detergent, respectively. Wells were scored as positive
for CTL recognition only if the level of specific lysis was more than
10%. Frequency values were determined with 95% confidence limits from
the cell input number at which 37% of the wells were negative for
recognition of the targets using the method of maximum likelihood,
fulfilling single hit kinetics by  2 analysis.
For bulk CTL lysis, stimulators, feeders, and effectors were treated as described for the limiting dilution assay, except that the concentrations for stimulators and effectors were 5 × 104 and 2.5 × 105, respectively. On day 14, the target cells were washed, and the effectors were harvested and plated at E (effector):T (target) ratio 20:1, 10:1, and 5:1 in triplicate. The plates were then incubated for 4 hours, harvested, and counted in the gamma counter. The percentage of specific cytotoxicity was calculated as (experimental release minus spontaneous release) / (maximum release minus spontaneous release) times 100. The mean ± SE levels of CTLp's for B-LCL infected with the control VSC11 were 17 ± 3/106 PBMCs and 7.6% ± 0.7% (E:T, 20:1), 5.8% ± 0.5% (E:T, 10:1), and 3.7% ± 0.3% (E:T, 5:1) for bulk CTL lysis (n = 185). Results for both CTLp's and the bulk CTL assays are presented as percentage virus-specific lysis, which equals percent specific lysis against the HHV-8 antigen-expressing targets minus percent specific lysis against the mock antigen-expressing targets. Single-cell IFN- , nitrocellulose-bottomed
96-well plates (Millipore, Bedford, MA) were precoated with 10 µg/mL
of an anti-IFN- monoclonal antibody (mAb), 1-DIK (Mabtech, Decatur,
GA) overnight at 4°C. The coated plates were washed 4 times with PBS
to remove unbound Ab and then blocked for 2 hours with RPMI 1640 supplemented with 10% human serum. Frozen PBMCs were thawed and added
to the wells with the autologous, infected B-LCL at
effectors/stimulators ratio of 2:1 and incubated overnight at 37°C
and 5% CO2. The cells were discarded the following day by
washing the plates 6 times with PBS containing 0.05% Tween 20, and the
second biotinylated anti-IFN- mAb, 7-B6-1 biotin (Mabtech), was
added at 2 µg/mL and incubated for 2 hours at 37°C in 5%
CO2. The plates were further washed 6 times with PBS/0.05% Tween 20, followed by addition of avidin horseradish
peroxidase-conjugated streptavidin (Vectastain Elite kit, Vector
Laboratories, Burlingame, CA) to the wells and incubation for 1 hour at
room temperature. The plates were washed 3 times with PBS/0.05% Tween
20 and 3 times with PBS. The spots were developed by addition of
3-amino-9-ethyl-carbazole (Sigma) diluted in 0.1 M acetate buffer, pH
5, containing H2O2. Reactions were allowed for
about 5 minutes and were stopped by washing under running tap water.
After drying, spots were counted using a dissection microscope. The
results were expressed as the number of spots per 106 PBMCs
or CD8+ T cells. Stimulation with phorbol myristate acetate
(PMA) and ionomycin (1 ng/mL and 1 µM, respectively) was used
as a positive control, and stimulation with medium and mock vaccinia
virus-infected B-LCL was used as a negative control.
The mean ± SE level of IFN- Blocking assay using HLA class I and class II antibodies Targets and stimulators were washed and suspended at a concentration of 1 × 106 cells/mL. The mAbs against HLA class I (W6/32, Accurate Chemical, Westbury, NY) or HLA class II (I3, Beckman-Coulter, Fullerton, CA) were added at a production assay, and incubated at 37°C
in 5% CO2 for 45 minutes. The remainder of the experiments
were performed as described above.
CD4+ and CD8+ T-cell enrichment Immunomagnetic beads specific for CD4 and CD8 (M-450, Dynal, Lake Success, NY) were used for negative selection. Frozen PBMCs (10 × 106) were thawed and mixed with either CD4 or CD8 beads for 1 hour at 4°C in a 2-mL plastic tube (Nalgene, Rochester, NY). The tube was then placed on a magnet for 3 to 5 minutes, the supernatants containing either CD8+-enriched or CD4+-enriched T cells were removed. The T cells enriched with CD4+ or CD8+ were used as effectors in both the bulk CTL and the single-cell IFN- production assays.
T-cell phenotyping For immunophenotyping, frozen PBMCs were thawed and washed in Hanks balanced salt solution supplemented with 2% FCS and 0.1% NaN3 and incubated (30 minutes at 4°C) with one of the following mAbs: FITC- or phycoerythrin (PE)-conjugated mAbs specific for CD3, CD4, CD14, CD38, CD28, HLA-DR, or CD45 (Becton Dickinson); CD45RA and CD45RO (Immunotech, Westbrook, ME); and PE-Cy*5 conjugated anti-CD4 and anti-CD8 mAb (Immunotech). After incubation, the PBMCs were washed with PBS and fixed with 1% paraformaldehyde. Surface expression was analyzed using an EPICS Elite flow cytometer (Beckman-Coulter).Statistical analysis The Fisher exact test was used for comparison of clinical signs and symptoms. The 2-tailed, paired Student t test was used for comparing levels of IFN- production and CTLp's. Nonparametric correlations of CTLp's and IFN--producing cells were assessed using the 2-tailed Spearman rank correlation test. The 3-way repeated measures ANOVA was used for pattern analysis.
Incidence and clinical signs and symptoms of primary HHV-8 infection We chose to assess the clinical and laboratory components of primary HHV-8 infection in HIV-1-negative persons in the MACS to avoid potential complications of underlying HIV-1 infection. In this group of homosexual men persistently seronegative for HIV-1, the HHV-8 seroprevalence rate was 9.2% (11 of 119) at study entry in 1984-1985, which is similar to the seroprevalence reported in the general population.1 From April 1984 through March 1999, 6 of the 108 HHV-8 seronegative men seroconverted to HHV-8. One of the 6 HHV-8 seroconverters was excluded from this investigation because of insufficient numbers of interim clinic visits to determine the time of seroconversion.As of March 1999, the overall incidence rate for HHV-8 infection was 3.7/1000 person-years (5 of 1361). However, the incidence was not constant over the years. These HHV-8 seroconversions occurred earlier in the study with the incidence rates being 10.1, 19.9, 9.8, and 9.8/1000 person-years in 1985, 1986, 1990, and 1991, respectively, with no subsequent HHV-8 seroconversions after 1991. No specific, severe illness was associated with primary HHV-8 infection
as noted in the extensive clinical assessment taken at the time of the
study subjects' clinic visits. However, several nonspecific, mild
clinical signs and symptoms were documented near the time of
seroconversion in 4 of the 5 men: mild, transient cervical and
submental lymphadenopathy and diarrhea in subjects 20 and 22 (Figure
1A,C), fatigue and localized rash on the
right ankle in subject 21 (Figure 1B), and cervical and submental
lymphadenopathy and localized facial rash in subject 24 (Figure 1E). No
signs and symptoms were associated with seroconversion in subject 23 (Figure 1D). To determine whether these signs and symptoms were significantly associated with HHV-8 seroconversion, we compared these
findings from the first seropositive visit for the HHV-8 seroconverters
with the same clinic visits in the 102 men who remained HHV-8
seronegative; 80% of the HHV-8 seroconverters reported at least one
symptom compared to 31.4% of the seronegative men (P = .043).
Longitudinal analysis of T-cell phenotypes in primary HHV-8 infection We assessed T-cell phenotypes, including memory and activation cell markers, to determine whether there were any changes during primary HHV-8 infection. This is because expansion of activated CD8+ T cells is a classic finding in EBV mononucleosis of adults.2-4 No changes were seen in T-cell numbers expressing lineage CD3, CD4, and CD8 molecules, naïve/memory cell markers CD45RA and CD45RO, or activation and costimulatory markers CD38, CD28, and HLA-DR, after primary infection with HHV-8 (Figure 2).
Effects of HHV-8 infection on CD8+ T-cell reactivity to CMV and PMA/ionomycin The immunosuppressive effects of primary HHV-8 infection were analyzed in the HHV-8 seroconverters by assessing the numbers of CD8+ T cells responding to a specific recall antigen and a mitogen. There were no significant changes in the numbers of IFN--producing cells either to the CMV pp65 matrix protein, which
is a major CTL target,32,33 or to the mitogen
PMA/ionomycin, after seroconversion to HHV-8 (Figure
3). Thus, primary HHV-8 infection differs
from primary symptomatic EBV infection that is classically associated with transient immunosuppression.5-7
Anti-HHV-8 CTLp's and IFN- -producing T cells to at least 3 of the 5 HHV-8 lytic cycle proteins concurrent with the appearance of
serum antibodies to the virus and viral DNA in the PBMCs (Figure 1A-E).
Bulk CTL responses specific for the HHV-8 lytic cycle proteins also
developed coincident with seroconversion and DNAemia, similar to the
CTLp responses (data not shown). In 3 of the 5 cases (Figure 1A,B,D), there was evidence of HHV-8 DNA in the blood approximately 2 to 9 months before the estimated time of seroconversion and increase in
titers of HHV-8-specific Ab. Notably, in subject 21, anti-HHV-8 T-cell
IFN- responses were detected concurrently with HHV-8 DNA at 3 and 9 months before seroconversion (eg, 280 spots and 340/106
PBMCs for gB, and 350 and 420/106 PBMCs for IE protein,
respectively; Figure 1B). This indicates that in some primary HHV-8
infections, DNAemia precedes production of detectable anti-HHV-8 serum
antibodies but is usually coincident with appearance of
CD8+ T-cell immunity to HHV-8.
The frequencies of CTLp's and IFN-
Analysis of these data by a 3-way repeated measures ANOVA for the
limiting dilution and single-cell IFN-
The frequencies of HHV-8 specific, IFN-
This study provides the first description of the natural history of primary HHV-8 infection in healthy adults negative for HIV-1. We identified a relatively low prevalence of HHV-8 infection (9.2%) at entry into the MACS in 1984-1985, and an overall incidence rate for HHV-8 infection of 3.7/1000 person-years between 1984 and 1999, which is similar to the recent report of primary HHV-8 seroconversions among HIV-1 uninfected homosexual men in an Amsterdam study.34 These rates, along with a decreasing trend in incident HHV-8 infection by calendar year, were not unexpected in our cohort of men persistently negative for HIV-1. HHV-8 seroprevalence in these men was similar to that in the general adult population (0%-8%),1 and lower than the 30% to 37% seroprevalence noted at study entry in HIV-1-infected homosexual men in the MACS.35,36 In this regard, we have observed a dramatic decrease in HIV-1 seroconversion in the MACS.37 Thus, under the hypothesis that among homosexual men, the same sexual behaviors are risk factors for HIV-1 and HHV-8 infection, a similar temporal decline in HHV-8 infection should be observed. There was no specific, severe illness associated with primary HHV-8 infection. However, transient, mild signs and symptoms of diarrhea, fatigue, localized rash, and cervical and submental lymphadenopathy were noted near the time of seroconversion to HHV-8 in 4 of the 5 seroconverters. This significantly differed from clinical findings in the 102 men who remained HHV-8 seronegative. Notably, the ratio of symptomatic to asymptomatic primary infections for the other human gammaherpesvirus, EBV, ranges from 3:1 to 1:10 in different adult populations.8 Thus, although HHV-8 does not appear to be a common cause of a severe, primary clinical syndrome in normal adults, it may be related to a mild syndrome that will require confirmation in larger studies of primary HHV-8 infection. Our finding that there were no significant alterations in numbers of
T-cell phenotypes or activated T cells during primary HHV-8 infection
also differs from primary, symptomatic EBV infection in adults, which
has a prolonged increase in the numbers of
CD8+CD45RO+ HLA-DR+ T
cells.2-4 Moreover, we did not find a suppressive effect
of HHV-8 primary infection on either CMV-specific or mitogen-stimulated CD8+ T-cell IFN- Of importance is that we documented a distinct, persistent HLA class I
restricted, CD8+ cell cytolytic and IFN- Lytic cycle proteins were also immunogenic for the humoral Ab system,
because Ab titers to HHV-8 lytic cycle proteins paralleled the T-cell
responses. A high prevalence of antibodies for other HHV-8 capsid
proteins has been detected in HHV-8 seropositive individuals,41,42 supporting the immunogenicity of lytic
cycle proteins of the virus. A viremic (DNAemia) phase appeared 2, 3, and 9 months prior to the appearance of detectable humoral Ab responses
to HHV-8 in 3 subjects and occurred coincident or after seroconversion
in the other 2 subjects. This may be related in part to variation in
the actual time of initial HHV-8 infection, which was at some point
within the approximately 6-month interval between the last HHV-8
Ab-negative and first HHV-8 Ab-positive results. Of interest is that
the earliest DNAemia, found at both 9 and 3 months before
seroconversion in one subject, was coincident with the appearance of
anti-HHV-8 IFN- The early presence of DNAemia in some of our individuals is similar to the recent Amsterdam study, which found HHV-8 DNA in the serum of 2 of their 7 cases of primary HHV-8 infections in HIV-1 negative persons 3 months prior to the determined date of seroconversion.34 Interestingly, 6 of their 11 HIV-1-infected participants also had HHV-8 DNA-positive sera up to 2 years before seroconversion, suggesting that there may be higher levels of HHV-8 DNAemia in HIV-1-infected persons before the presence of detectable Ab. However, our results contrast with the Amsterdam study in that we detected viral DNA in PBMCs for several years after seroconversion, whereas they detected viremia in the serum for only a few months after seroconversion. This discrepancy may be due to a lower viral DNA copy number in serum compared to PBMCs as the infection evolves into a persistent, latent state. The sporadic, HHV-8 DNA-negative samples in 2 of our 5 subjects after seroconversion may be related to relatively low levels of this DNA in their peripheral blood, as has been reported for some healthy, HHV-8-seropositive, HIV-1-negative individuals.44 The levels of CD8+ T-cell reactivity to most of the HHV-8 lytic cycle proteins declined several years after primary infection. This may be related to a decrease in lytic replication of the virus and resultant lower antigenic load. This could also explain why relatively low CTL reactivity to HHV-8 lytic cycle proteins has been found in cross-sectional studies of persons with unknown duration of HHV-8 infection.23,24 Indeed, viral DNA persisted in the PBMCs for at least 2 years after seroconversion in the 5 subjects in this study, suggesting an establishment of chronic, latent HHV-8 infection. CD8+ T-cell responses to HHV-8 could be operative in controlling reactivation of virus in this latent phase of HHV-8 infection. As recently reported for other viral infections,45 we
found that the number of cells producing IFN- Our results support the role of CD8+ T cells specific for HHV-8 lytic proteins in control of primary HHV-8 infection. Recent cross-sectional studies found lower anti-HHV-8 CTL23,24 and CD4+ T-cell responses in HHV-8-seropositive, HIV-1-infected persons.47 Longitudinal studies are needed to delineate the function of HHV-8-specific T-cell reactivity in the development of HHV-8-associated diseases such as KS during HIV-1 infection and immunosuppressive drug treatment in organ and tissue transplant recipients.
This study was part of the requirements for MS and PhD degrees by Q.J.W. in the Department of Infectious Diseases and Microbiology of the University of Pittsburgh Graduate School of Public Health. We thank Dr Xiao-Li Huang and Dr Zheng Fan for research suggestions; Laurie Johnson, Christine Kalinyak, Xiao Mao, Luann Borowski, Susan McQuiston, Gayle Springer, and Bill Buchanan for technical and clinical assistance; and the Pitt Men's Study MACS staff and volunteers for their dedication and support.
Submitted November 17, 2000; accepted December 15, 2000.
Supported in part by grants from the US Public Health Service (P30 CA47904, R01 CA82053, R01 CA75957, R03 CA81600 and U01 AI35041).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Charles R. Rinaldo Jr, Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, A427 Crabtree Hall, 130 De Soto St, Pittsburgh, PA 15261; e-mail: rinaldo+{at}pitt.edu.
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Abstract
Introduction
20°C. For each assay, the fixed cells were first
incubated in PBS containing 10% goat serum for 1 hour at 37°C to
block nonspecific binding. The blocking buffer was removed and primary
Ab (diluted in PBS containing 10% goat serum) was added. The slides
were incubated for 1 hour at 37°C. Two dilutions (1:50 and 1:100) of
primary Ab (participant's sera) were tested for each serum sample. The
cells were next washed extensively in PBS, treated with the secondary
Ab (fluorescein isothiocyanate [FITC]-goat antihuman IgG diluted
1:200 in PBS) and incubated at 37°C for 0.5 to 1 hour. The cells were
again washed extensively in PBS and glass coverslips added. For each assay, we confirmed the specificity of the fluorescence using cells
incubated with primary but no secondary Ab, and cells incubated with
secondary but no primary Ab. For each batch of serum samples tested,
known HHV-8 positive and negative sera were included. All serum samples
were tested twice in a blinded fashion and assessed microscopically by
the same reader. Assays using the induced BCBL-1 cells were examined
for the presence of whole cell immunofluorescence. The serum samples
were serially diluted to determine the Ab titers (reciprocal of the
last positive dilution). The cutoff titer for positive reactivity
was 50.
; to PMA/ionomycin, 
. The data are the mean ± SE for at least 3 individuals at each time point. SC represents the
estimated time of seroconversion to HHV-8 in these individuals.
) and MiCP
(
) by anti-HLA class I but not class II in representative subject
14. (B) Blockage of HHV-8-specific IFN-
) by anti-HLA class I but not class
II Abs in representative subject 21. (C) HHV-8 specific CTL responses
to gH (
, gB; 
, MCP; 
, MiCP; 
,
IE.