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Journal of Virology, April 2000, p. 3478-3485, Vol. 74, No. 8
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Identification of Antigenic Proteins Encoded by Human Herpesvirus
8 and Seroprevalence in the General Population and among Patients
with and without Kaposi's Sarcoma
Harutaka
Katano,1
Takuya
Iwasaki,1
Nobuyoshi
Baba,1
Masanori
Terai,1
Shigeo
Mori,2
Aikichi
Iwamoto,3
Takeshi
Kurata,1 and
Tetsutaro
Sata1,4,*
Department of
Pathology1 and Laboratory of
Pathology,4 AIDS Research Center, National
Institute of Infectious Diseases, Shinjuku, Tokyo 162-8640, and
Departments of Pathology2 and
Infectious Diseases,3 Institute of
Medical Science, University of Tokyo, Minato, Tokyo 108-8639, Japan
Received 15 November 1999/Accepted 19 January 2000
 |
ABSTRACT |
To establish a sensitive and specific antibody assay, potent
antigenic proteins encoded by human herpesvirus 8 (HHV8) were studied.
Fifteen recombinant HHV8-encoded proteins were produced as glutathione
S-transferase fusion proteins. The sera from
AIDS-associated Kaposi's sarcoma (KS) patients reacted with four
proteins encoded by open reading frames (ORFs) K8.1, 59, 65, and 73 in
a Western blot assay. An enzyme-linked immunosorbent assay (ELISA)
using these four proteins as antigens (mixed-antigen ELISA) revealed that all 26 sera derived from KS patients (24 with and 2 without human
immunodeficiency virus infection) became positive for anti-HHV8 antibodies. The presence of HHV8 was demonstrated in 14 (1.4%) of
1,004 sera from the Japanese general population and 10 (1.9%) of
527 sera from patients without HHV8-associated diseases. The presence
of immunoglobulin G (IgG) and IgM antibodies against HHV8 examined
further by the mixed-antigen ELISA and Western blotting revealed
IgG antibody in all ELISA-positive sera, while IgM antibody against ORF
K8.1 was absent. These data suggest that the ORF 73 and 65 proteins are
potent antigens for a sensitive serological assay.
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INTRODUCTION |
The human herpesvirus 8 (HHV8,
Kaposi's sarcoma [KS]-associated herpesvirus) DNA sequence has been
demonstrated in all forms of KS, primary effusion lymphomas (PEL, body
cavity-based lymphomas [BCBL]), and a subset of patients with
multicentric Castleman's disease (MCD) (4, 18, 19, 28). For
a serological survey, PEL cell lines have been employed for
immunofluorescence assay (IFA) and Western blot analysis to demonstrate
antibodies against HHV8 in patients' sera (9, 13, 26). By
Western blot analysis, sera from KS patients reacted with many proteins
encoded by HHV8 open reading frames (ORFs) (1-3, 6, 9, 14,
20-23), of which more than 80 are known to be present in the
HHV8 DNA (25). However, little information about the
predominant antigen is available. By immunoscreening of a cDNA library
from 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced
BCBL-1 cells using a human immunodeficiency virus-positive
(HIV+), KS+ serum, 12 ORF proteins (encoded by
ORFs 6, 8, 9, 25, 26, 39, 59, 65, 68, 73, K8.1A, and K8.1B) were
identified as the major proteins (3). Among these, the ORF65
protein (minor capsid protein) has been determined to be the most
potent antigen for serological examination of KS sera (15,
20). The K8.1 protein has been cloned and characterized and has
also been shown to be one of the predominant and useful antigens for
serological analysis (2, 14, 21). Recently, it was shown
that lytic (ORFs 65, K8.1A, and K8.1B) and latent (ORF73) proteins
exhibited high reactivity with the sera from HHV8-seropositive
individuals on Western blot analysis (30).
To date, anti-HHV8 antibodies in human sera have been detected by two
methods, IFA and enzyme-linked immunosorbent assay (ELISA) (1, 5, 6, 8, 13, 16, 17, 20, 22, 24, 26). The target
antigens of the two methods differ. Latency-associated nuclear antigen (LANA) derived from HHV8-encoded ORF73 and
TPA-induced antigens in PEL cell lines have been employed for IFA,
while the ORF65 and ORF26 proteins (possible minor capsid protein) or
lysates of whole virus particles derived from KS-1 cells have been used as antigens for ELISA (1, 5, 6, 8, 13, 16, 17, 20, 22, 24,
26). The use of these different antigens often results in
discrepancy between the data obtained from these serologic assays.
Current HHV8 antibody testing, therefore, is of uncertain accuracy
mainly in patients with asymptomatic HHV8 infection (22), and the definitive seroprevalence of HHV8 infection in specific populations cannot yet be determined clearly.
Despite the uncertain accuracy of serological tests for HHV8,
several groups have reported the seroprevalence of antibodies to
HHV8 in the general population. The positivity rates in these studies
have varied from 0 to 53%, depending on the assay methods used
and the countries examined (5, 8, 16, 29). Infection with
HHV8 seems to be uncommon in the United States (0 to 25%) and
Northern Europe (3 to 5.1%), more common in certain Mediterranean countries (4 to 12%), and widespread in countries such as Uganda in
Africa (35 to 53%). The transmission modes of HHV8 have also not been
clarified yet. In countries with endemic infection, horizontal transmission among children has been suggested to be prevalent (16) while sexual transmission appears to play an important role particularly among homosexual men in countries with nonendemic infection.
In this study, the predominant antigenic proteins encoded by HHV8 in
AIDS-KS patients were investigated by the use of recombinant glutathione S-transferase (GST) fusion proteins and we have
succeeded in developing a highly sensitive ELISA system for the
detection of anti-HHV8 antibodies. In addition, HHV8 seroprevalence in
the Japanese general population and among patients with various
diseases was examined using this ELISA system.
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MATERIALS AND METHODS |
Recombinant HHV8 ORF proteins.
Fifteen sequences in the
published HHV8 ORFs (25) amplified by PCR using the primers
listed in Table 1 were cloned into bacterial expression vectors pGEX5X-2 (Pharmacia, Uppsala, Sweden) and
pGEX2T (only ORF65; Pharmacia) at the two restriction enzyme sites. The
selection of these proteins was based on our unpublished results of the
immunoscreening of a cDNA library from TY-1 cells using sera of
patients with AIDS-KS and on the published data of Chandran et al.
(3, 10, 11). Among these proteins, K13, ORF72, and ORF73 are
thought to be latent proteins and the others are thought to be lytic
ones (7). The sequences of K2, ORF26, K8, K11, ORF65, K13,
ORF72, and K14 were cloned almost in full length, whereas partial
sequences of the proteins of ORF6, K10, ORF59, ORF64, and ORF73 were
cloned for convenience of amplification and expression. For the cloning
of ORF73, two partial sequences of the proteins, i.e., the amino- and
carboxy-terminal parts, were cloned and these proteins were
designated ORF73N and ORF73C, respectively. The constructs for protein
expression formed fusion proteins consisting of GST and each of
the ORF proteins. These were then expressed in Escherichia
coli and affinity purified using glutathione-Sepharose as
described previously (27). The purity and concentration of
the eluted proteins were assessed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and the Bradford assay
(Protein Assay; Bio-Rad, New York, N.Y.), respectively.
Western blot analysis and IFA.
The Western blot analysis was
carried out using sera from four AIDS-KS patients, one AIDS-PEL
patient, one HIV-negative KS patient, one Epstein-Barr virus
(EBV)-positive patient with infectious mononucleosis, and a healthy
Japanese blood donor. All sera were diluted 1:200 in Block Ace
(Snowbrand Milk Products, Tokyo, Japan). Cell lysates of a TPA-induced
TY-1 cell line (HHV-8 positive, EBV negative) and recombinant HHV8 ORF
proteins were used for the Western blot analysis (10).
Briefly, TY-1 cells were grown in RPMI medium supplemented with 10%
fetal calf serum. After the addition of TPA at 20 ng/ml to the medium,
the TY-1 cells were cultured for 2 days. After being washed two times
in phosphate-buffered saline (PBS), 107 TPA-induced TY-1
cells were lysed in 1 ml of sample buffer consisting of 25 mM Tris-HCl
(pH 8.0), 5% glycerol, 1% sodium dodecyl sulfate, 1%
2-mercaptoethanol, and 0.05% bromophenol blue. For the cell lysate and
recombinant HHV8 ORF proteins in the sampling buffer, 5 µl per lane,
were applied to 10% polyacrylamide gels and then electrotransferred to
membranes (Immobilon; Millipore, Bedford, Mass.). After blocking with
Block Ace, the diluted serum samples were allowed to react for 60 min
at room temperature. Goat anti-human immunoglobulin antibody conjugated
with alkaline phosphatase (code no. AHI0705; Tago Immunologicals,
Camarillo, Calif.) was used as the secondary antibody. To confirm the
presence of GST fusion proteins on the membranes, a rabbit
affinity-purified anti-GST polyclonal antibody and goat anti-rabbit
immunoglobulin G (IgG) conjugated with alkaline phosphatase (Tago
Immunologicals) were used as the primary and secondary antibodies,
respectively. Furthermore, to identify IgG and IgM antibodies against
HHV8 in the ELISA-positive sera, Western blot analysis using specific
antibodies against human IgG or IgM (code no. 4600 and 2492; Tago
Immunologicals) as the secondary antibodies was performed as described above.
The IFA was carried out as described previously (
13,
26). In
brief, TPA-induced TY-1 cells were spotted onto a slide (Erie
Scientific Co., Erie, Colo.) after being washed two times in PBS
and
dried and then fixed in acetone for 10 min at room temperature.
The
patients' sera were diluted 1:40 in PBS-2% fetal calf serum
and
applied to the slide for 45 min at room temperature. Rabbit
anti-human
IgG conjugated with fluorescein isothiocyanate (Tago
Immunologicals)
was used as the secondary antibody. Between these
steps, the slides
were washed three times each in PBS for 5 min.
The positive reaction of
HHV8 antibody by IFA was determined by
the presence of LANA, other
nuclear antigens, and/or cytoplasmic
antigens.
Sera.
Informed consent was obtained from all of the patients
with HIV infection and other diseases, and from the healthy donors, before blood samples were obtained. Sera obtained from 21 HIV+ KS+ adult male patients and 17 HIV
KS healthy donors were tested further
to confirm the sensitivity and specificity of the various ELISAs.
Moreover, 20 sera of healthy donors who were confirmed to have
anti-cytomegalovirus (CMV) antibodies by CMV ELISA and 16 sera of
patients with infectious mononucleosis who had been confirmed to be
positive by both EBV ELISA and IFA were also tested by our
mixed-antigen ELISA. We tested the sera of 1,004 healthy Japanese
donors which were received from the World Health Organization and the
National Serum Reference Bank/Tokyo, National Institute of Infectious
Diseases (http://idsc.nih.go.jp/yosoku99/index-E.html). These sera were
collected from all districts in Japan and from all generations equally
in order to survey the prevalence of various infectious diseases. Sera
of 527 patients with various diseases were also investigated (for the
underlying diseases, see Table 3). All sera were stored at 
20°C and
heat inactivated at 56°C for 30 min before use.
ELISA using recombinant HHV8 ORF proteins.
Purified
recombinant GST fusion proteins (2 µg/ml, each protein) diluted in
100 mM carbonate buffer, pH 9.0, were used to coat the wells of ELISA
plates (50 µl per well; Corning Coaster 3690 enzyme
immunoassay-radioimmunoassay plate; Corning Glass Works, Corning, N.Y.)
overnight at 4°C. After being washed in washing buffer (0.1 M PBS
[pH 7.4], 0.02% Tween 20), the serum or plasma samples at various
dilutions in the dilution buffer (Block Ace) were allowed to react for
30 min at 37°C with the recombinant proteins. To avoid nonspecific
reactions with GST, the lysate of E. coli producing
recombinant GST protein was added to the dilution buffer at a
concentration of 500 µg/ml. Unbound serum was removed by the washing
buffer. Mixed goat anti-human immunoglobulins (Igs, including IgG, IgA,
IgM, and Ig light chains) conjugated with alkaline phosphatase (code
no. AHI0705; Tago Immunologicals) were then added, and incubation was
conducted for 30 min at 37°C. After washing, phosphate substrate
tablets (5 mg per tablet; Sigma, St. Louis, Mo.) were used as the
substrate to develop the color for 30 min. The absorbance (Ab) of the
wells was measured at a wavelength of 405 nm. In the mixed-antigen
ELISA, five recombinant proteins, i.e., K8.1, ORF59, ORF65, ORF73N, and
ORF73C, were mixed at 2 µg/ml per protein and used to coat the wells
at 50 µl per well. In an ELISA specific for IgG or IgM antibody, goat
anti-human IgG or IgM conjugated with alkaline phosphatase (code no.
4600 and 2492; Tago Immunologicals) was used as the secondary antibody. The specificity of anti-human IgG and IgM was guaranteed by the supplier.
Based on the surveys of our negative control sera and sera from
patients with AIDS-KS, an ELISA titer of 1:100 was considered
to be
positive. This dilution was chosen to evaluate the final
results of the
mixed-antigen ELISA by considering the positivity
of these sera for
LANA by IFA using HHV8-positive PEL cells. The
cutoff value for the
mixed-antigen ELISA was determined by the
mean Ab plus 5 standard
deviations (SD) for 43 normal serum samples.
Different cutoff values
were calculated among ELISAs for mixed
Igs, IgG, and IgM. Only for the
data shown in Table
2 was the
cutoff value determined by the mean Ab
plus 3 SD because it represents
a comparison of proteins. To avoid
different values between plates,
two serum samples from an AIDS-KS
patient and a healthy donor
were placed as positive and negative
controls on every plate.
Moreover, all calculations were based on the
values calculated
as follows: (sample Ab
negative control
Ab)/(positive control
Ab
negative control Ab). For statistical
analysis, a two-sample
t test was
employed.
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RESULTS |
Different reaction patterns of sera from AIDS-KS patients.
In
order to confirm the sizes of antigenic proteins encoded by HHV8, the
reactivity of the sera of AIDS-KS patients with lysates of the
HHV8-infected PEL cell line TY-1 was first examined by Western blot
analysis. Several bands, including the 222- and 234-kDa bands of LANA
and the 50-kDa band of ORF59, were noted (Fig.
1), and the reaction patterns varied, but
in general, the patterns were similar to those reported before
(23). The variation of these patterns depended on the sera
examined, and no band was found for the sera of patients with
infectious mononucleosis and healthy individuals. These data showed
clearly that many antigenic proteins encoded by HHV8 existed in the
lysate of the TPA-induced TY-1 cell line and that every serum sample
recognized these in different patterns.
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FIG. 1.
Western blot analysis using TPA-induced TY-1 cell
lysates revealed that the sera of AIDS-KS or PEL patients reacted with
many bands and that the reaction patterns varied depending on the
individual sera (lanes 1 to 6). No band was detected in the lanes
containing the sera from a patient with infectious mononucleosis (IM)
and a healthy blood donor (lanes 7 and 8). The reported sizes of the
LANA and ORF59 bands are indicated on the right in kilodaltons.
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To clarify the antigenic proteins encoded by HHV8, we prepared 15 recombinant proteins using the GST fusion protein system,
i.e.,
for ORF6, K2, ORF26, K8, K8.1, K10, K11, ORF59, ORF64, ORF65,
K13,
ORF72, ORF73N, ORF73C, and K14. Western blot analysis indicated
that the sera from patients with KS or PEL reacted with some of
these
recombinant proteins, i.e., the K8.1, ORF59, ORF65, ORF73N,
and ORF73C
proteins; however, the reaction patterns differed for
each serum sample
(Fig.
2). The patterns were classified
into
the following three categories; (i) strong reaction with both
lytic (ORF26, ORF59, and ORF65, etc.) and latent (ORF73) proteins
(sera
1 and 2 in Fig.
2), (ii) strong reaction with lytic proteins
but weak
reaction with latent proteins (sera 3 and 4), and (iii)
weak reaction
with lytic proteins but strong reaction with latent
proteins (serum 6).
No antigen was found to react with all of
the sera examined. None of
the sera recognized the ORF6, K2, K8,
K10, ORF64, K13, ORF72, or K14
protein.
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FIG. 2.
Western blot analysis. Some of the recombinant GST
fusion proteins were recognized by AIDS-KS or PEL patients' sera. The
sera in lanes 1 to 8 are the same as those in Fig. 1. Only the
recombinant protein bands in the membrane, whose sizes are listed in
Table 1, are shown. IFA results are indicated at the bottom. The K8.1,
ORF59, ORF65, and ORF73 proteins were recognized by some sera.
Reactions with other proteins were weak. Lane 9 indicates that each
protein was present on the membrane. IM, infectious mononucleosis.
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Identification of antigenic proteins for ELISA.
Considering
the variable reaction patterns among the sera in the Western blot
analysis, the K8.1, ORF59, ORF65, ORF73N, and ORF73C proteins
were selected as candidate antigens for establishing an ELISA system to
detect anti-HHV8 antibodies. Twenty-one AIDS-KS and 17 normal serum
samples were tested by ELISA using each of the proteins as the antigen.
All of these 21 AIDS-KS serum samples were confirmed to be positive by
IFA. As shown in Fig. 3 and Table 2, the sera from some of these
AIDS-KS patients reacted with the K8.1, ORF59, ORF65, ORF73N, and
ORF73C proteins in the ELISA. However, each ELISA system using only one
protein as the antigen missed some of the anti-HHV8 antibodies present
in the AIDS-KS sera. Both ORF73 proteins reacted with over 80% of the
AIDS-KS serum samples. Three serum samples found to be negative by
ELISA using the ORF73 protein as the antigen reacted with the K8.1 or ORF65 protein. In the ELISA using ORF59, there was no significant difference between the optical density values of AIDS-KS and normal sera overall but four serum samples reacted with the ORF59 protein and
also reacted strongly with both lytic and latent proteins. All AIDS-KS
serum samples were finally found to be positive by ELISA using all of
these five recombinant proteins as mixed antigens. Therefore, we
established a highly sensitive ELISA system for detecting anti-HHV8
antibodies using mixed antigens comprising the ORF59, ORF65, K8.1,
ORF73N, and ORF73C proteins and designated it a mixed-antigen ELISA.
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FIG. 3.
Group scatter diagrams of ELISAs using the recombinant
proteins. The scatter diagrams show reactions with various recombinant
proteins in each ELISA. Compared with normal sera (right), AIDS-KS sera
(left) reacted with the K8.1, ORF59, ORF65, ORF73N, ORF73C, and mixed
proteins in ELISAs. The vertical axis indicates Ab, and the means and
SD are shown on the right.
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To check the specificity of the mixed-antigen ELISA, the reactivity and
titers of the sera used in a previous experiment were
tested again by
IFA and Western blot analysis using HHV8-positive
cell lines (TY-1 and
BCBL-1; data not shown). The results of IFA
and Western blot analysis
were in accordance with those of the
mixed-antigen ELISA. Moreover, 20 serum samples from CMV-infected
individuals and 16 from patients
with infectious mononucleosis
were all found to be negative by the
mixed-antigen ELISA. These
data strongly suggested that no
cross-reactions with CMV and EBV
had occurred in this ELISA
(Table
3). Thus, it was concluded
that
the mixed-antigen ELISA was specific for HHV8.
Seroprevalence of anti-HHV8 antibodies in the Japanese general
population and in patients with various diseases.
Using this
mixed-antigen ELISA system, the seroprevalence of anti-HHV8 antibodies
in the Japanese general population and among patients with various
diseases was investigated (Table 3). The antibodies were found in 14 (1.4%) of 1,004 serum samples from the general population. All of the
mixed-antigen ELISA-positive sera were confirmed to be positive by IFA
and Western blot analysis using the recombinant proteins (data not
shown). The 14 HHV8 antibody-positive sera were from nine (64.3%)
males and five (35.7%) females (mean age, 26 years; age range, 0 to 66 years). Moreover, 5 of the 14 mixed-antigen ELISA-positive serum
samples from young individuals (mean age, 12 years; age range, 0 to 29 years; three males and two females), were positive for IgM antibodies
by the mixed-antigen ELISA using anti-human IgM antibody as the
secondary antibody, while the remaining 9 were positive for only IgG
antibodies to HHV8 (data not shown). The reactivity of IgM was
confirmed by Western blot analysis as described in the next section.
Our mixed-antigen ELISA also revealed that all of the 24 serum samples
from AIDS-KS patients were positive for anti-HHV8 antibodies.
The sera
of patients with MCD and AIDS patients who were homosexual
exhibited
high positivity rates (30.0 and 63.6%, respectively).
On the other
hand, 10 (1.9%) of the 527 sera from patients with
various diseases
other than the HHV8-associated diseases were
positive. All of these
sera were also confirmed to be positive
by IFA and Western blot
analysis using recombinant proteins (data
not shown). No significant
association between the underlying
diseases and HHV8 infection
was found. The mixed-antigen ELISA
specific for the IgM or IgG
antibody revealed that 7 of the 24
AIDS-KS sera were positive for both
IgM and IgG antibodies, while
the rest were positive for only the IgG
antibody (data not shown).
All of the 10 positive sera from patients
with various diseases
were negative for the IgM
antibody.
Reactivity of ELISA-positive sera with the K8.1, ORF59, ORF65, and
ORF73 proteins on Western blot analysis.
The Western blot analysis
for IgG or IgM antibody to HHV8 revealed that the IgG antibody was
present in all of the mixed-antigen ELISA-positive sera, while the IgM
antibody was found in 7 (30%) of the 23 sera from patients with KS
(six HIV+ and one HIV) and 5 (36%) of the 14 from the general population (0, 2, 6, 23, and 29 years old, three males
and two females; Table 4). All of the
ELISA-positive sera reacted mainly with the recombinant ORF65 and
ORF73N proteins, while the ORF59 and K8.1 proteins were recognized by
some sera. Interestingly, the K8.1 protein was recognized by the IgG,
but not the IgM, antibody. In addition, this protein reacted
predominantly with the sera from KS patients and rarely with the sera
from the general population or from patients with various diseases
other than the HHV8-associated diseases.
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DISCUSSION |
The present study revealed that (i) the reaction patterns of
AIDS-KS sera with HHV8-encoded proteins varied; (ii) the ORF59, ORF65,
K8.1, and ORF73 proteins were the most strongly antigenic for AIDS-KS
patients; (iii) the seroprevalence of HHV8 was 1.4% in the general
population and 1.9% among patients with various diseases in Japan; and
(iv) the K8.1 proteins were recognized predominantly by sera from KS
patients. A highly sensitive ELISA for detection of anti-HHV8
antibodies was also established.
Western blot analysis revealed that the reaction patterns of the
examined sera with the lysates of HHV8-infected cells were different.
We speculate that the variation in the reaction patterns arose from the
viral replication status, immune response, and the disease(s) of each
patient. In the present study, we selected the K8.1, ORF59, ORF65, and
ORF73 proteins as the antigens for the mixed-antigen ELISA system. This
selection was considered to be appropriate based on the evidence shown
in Fig. 2 and 3 and Table 2. This protein panel may not be sufficient
for detection of the entire spectrum of anti-HHV8 antibodies, because
some of the sera also reacted with other bands, as illustrated in Fig. 1. However, we concluded that these proteins are sufficient for the
detection of anti-HHV8 antibodies because of the high positivity rate
obtained with the AIDS-KS sera examined. Our selection of these
proteins partially corresponded to that reported by Zhu et al.
(30).
Among these proteins, ORF73 (LANA) is known to be a latent protein and
ORF65 and K8.1 are known to be lytic proteins (21, 23). The
effective antigenicity of ORF65 and K8.1 for antibody assay has also
been reported (1, 2, 6, 14, 20, 21, 22). Our K8.1 and ORF65
positivity rates were lower than those previously reported (2, 14,
15, 20, 21, 22). We speculate that there are two reasons for
this. One is that we employed the first exon of K8.1
protein, and
another reason is the difference in the samples. It may be important
that the antigens used for an anti-HHV8 antibody assay contain both
latent and lytic proteins, because some of the sera in this study
reacted only with either latent or lytic proteins. Our ELISA data (Fig.
3 and Table 2) suggest that LANA is recognized by most of the sera from
HHV8-infected individuals. Figure 3 and Table 2 also indicate that some
of the sera had antibodies only against latent proteins. Taken
together, these data suggest that antibodies against latent proteins
are the major components of anti-HHV8 antibodies in human sera and that
LANA exhibited stronger antigenicity than lytic proteins in the
antibody assay. In addition, we recently reported that most of the
spindle cells in KS express LANA in their nuclei (12). Therefore, previous ELISA systems employing one of the HHV8-encoded lytic proteins as an antigen were probably insufficient. Likewise, the
ELISA system using whole virus lysate as the antigens may miss the
antibodies against latent proteins. In fact, we observed that some of
the sera positive by our mixed-antigen ELISA were negative by an ELISA
using whole virus lysate as the antigens (Advanced Biotechnologies
Inc.) (data not shown; 5). Thus, it is essential for
the effective detection of anti-HHV8 antibodies in sera to employ both
latent and lytic proteins as antigens.
There are two reports on the prevalence of anti-HHV8 antibodies in
Japanese subjects. Fujii et al. reported finding that the seroprevalence of HHV8 infection among healthy Japanese blood donors
was 0.2% using IFA to detect LANA (8), and in our previous study, we reported that 2.2% of HIV-negative and KS-negative Japanese patients had antibodies against HHV8 as determined by ORF59 ELISA (11). Chatlynne et al. demonstrated that 11% of sera from
blood donors in the United States were positive for HHV8 antibodies by
a whole-virus lysate ELISA (5). This frequency is much
higher than that in the Japanese general population, and the
discrepancy may be attributed to the difference in race. The
seropositivity rate of anti-HHV8 antibodies was also found to be higher
in the elderly than in other age groups (Table 3). However, further analyses are required to determine the precise positivity rate in each
generation because the number of positive sera was limited in this study.
Our mixed-antigen ELISA system revealed that the positivity rate was
slightly higher in sera from patients with various diseases than in
those from the Japanese general population. This result suggests the
possibility that underlying diseases and as yet unknown factors may
activate HHV8 infection. However, we could not find any definite
association between HHV8 infection and any underlying disease. Sera
from patients with cerebral infarction (mean age, 83.6 years; age
range, 71 to 98 years) showed a relatively high positivity rate for
HHV8 antibodies (13.8%); however, this is unlikely to indicate any
association of HHV8 infection with cerebral infarction because of the
small number of samples examined.
Our data from Western blot analysis using anti-human IgG or IgM
antibody as the secondary antibody indicate that the K8.1 protein
reacts with only the IgG antibody from KS patients, suggesting an
association of the K8.1 protein with the pathogenesis of KS, although
further studies are necessary to verify the association. In the present
study, IgM antibodies in the sera from HHV8-infected individuals were
noted, for the first time, to recognize mainly the ORF73 and ORF65 proteins.
In conclusion, we demonstrated the antigenicity of HHV8-encoded
proteins in AIDS-KS patients and have developed a new sensitive ELISA
system. We believe that our mixed-antigen ELISA system will be a useful
tool in the clinical diagnosis of HHV8-related diseases and in
screening for and monitoring of antibodies against HHV8 in AIDS
patients and organ transplant recipients.
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ACKNOWLEDGMENTS |
We are very grateful to Y. Matsunaga and S. Inoue, Infectious
Disease Surveillance Center, National Institute of Infectious Diseases;
M. Goto, Department of Infectious Diseases, Institute of Medical
Science, University of Tokyo; N. Tachikawa and S. Oka, AIDS Clinical
Center, International Medical Center; G. Masuda, Department of
Infectious Diseases, Komagome Metropolitan Hospital; T. Kumasaka,
Department of Pathology, Juntendo University School of Medicine; H. Mizoguchi, Department of Hematology, and K. Yoda, Department of
Otorhinolaryngology, Tokyo Women Medical University; T. Minematsu,
Department of Microbiology, Miyazaki Medical College; N. Suematsu,
Department of Pathology, Yokufukai Hospital; A. Maeda, Department of
Pediatrics, Kochi Medical College; R. Muraki, Department of
Dermatology, National Kasumigaura Hospital; H. Harada, Department of
Dermatology, St. Lukes International Hospital; A. Urabe, Division of
Hematology, Kanto-Teishin Hospital; M. Ito, Department of Pediatrics, Mie University School of Medicine; and T. Ihara, Department of Pediatrics, Mie National Hospital, for providing the serum samples. We
thank B. Herndier, Department of Pathology, University of California, San Francisco, for supplying the BCBL-1 cell line.
This study was supported by Grants-in-Aid for Scientific Research from
the Ministry of Health and Welfare, Japan.
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FOOTNOTES |
*
Corresponding author. Mailing address: Laboratory of
Pathology, AIDS Research Center, and Department of Pathology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo 162-8640, Japan. Phone: 81-3-5285-1111, ext. 2627. Fax: 81-3-5285-1189. E-mail: tsata{at}nih.go.jp.
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Journal of Virology, April 2000, p. 3478-3485, Vol. 74, No. 8
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
