Bridging the Gap: Conference Record [Abstract book, International Conference on AIDS (12th: 1998: Geneva, Switzerland)]

534 Abstracts 31193-31196

12th World AIDS Conference

negative and EBV negative patient. For immunofluorescence assay we have
used fixed KS-1 lymphoma cells expressing >40% lytic antigens of HHV-8. The
EIA and IFA assays correlated well (p0.0001). The prevalence of HHV-8 antibodies varied in normal blood donors from USA (11%), Thailand (3%), Malaysia
(4.4%), India (3.7%) and Jamaica (15%). The finding that 40% of HIV-1 positive
individuals from Thailand were HHV-8 antibody positive in contrast to the HIV-1
negative subjects. There was a higher prevalence of HHV-8 antibodies found
in IVDU HIV-1 infected females (47%), in HIV-1 infected gay men (50%) and in
HIV-1 infected African females (80%), suggesting that these groups may be at
high risk for developing KS. Prevalence of HHV-8 antibodies in Kaposi's sarcoma
patients were 92% and the titers were also significantly elevated.
High antibody titers to HHV-8 were a good predictor for the development of
malignancies such as Kaposi's sarcoma or primary effusion lymphomas in the
US It would be important to see if the increase of malignancies in Southeast Asia
and Jamaica will be similar.
31193 HHV-8 Clades in Kaposi's sarcoma lesions: Phylogenetic
and phenetic analysis of variants from three continents
Franco M. Buonaguro, M.L. Tornesello, L. Buonaguro, M. Monaco,
E. Beth-Giraldo, G Giraldo. Viral Oncology, 1st. Naz. Tumori "F Pascale"
Cappella Cangiani, 1-80131 Naples, Italy
Objectives: To identify HHV-8 variants in KS lesions from several geographical
regions and their possible pathogenic role.
Methods: Since the detection of Herpesvirus-like particles in KS biopsies (G.
Giraldo et al., 1972) and the identification of the Human HerpesVirus 8 (HHV-8)
(Y. Chang et al., 1994) several variants of HHV-8 have been reported in Kaposi's
sarcoma (KS) and body-cavity-based lymphomas (BCBL). HHV-8 sequences,
detected in our laboratory in KS lesions from HIV-positive as well as HIV-negative
patients recruited in Europe (Greece and Italy), America (USA and Brazil) and
East Africa (Uganda and Kenya) (F.M. Buonaguro et al., 1996), have been analyzed by phylogenetic and phenetic analysis of the ORF26 (Minor Capsid Protein),
OPF72 (Cyclin D homolog) and K1. Full length ORFs have been amplified by
the appropriate oligoprimers; amplification products have been sequenced either
following a PEG-precipitation step (direct sequencing) or following subcloning in
pUC-based plasmid vectors. Multiple sequence alignments have been performed
with the MegAlign (DNASTAR Inc., Madison, WI, USA). Phylogenetic analysis has
been performed, using bootstrap analysis (100 bootstrap)and the neighbor-joining
method, with the software package TREECON (Y. Van de Peer, Univ. of Antwerp,
Belgium). Phenetic analysis and in particular synonymous/non-synonymous mutation rates have been calculated by the Nei and Gojobori method using the
software PAML (Z. Yang, Univ. of California, Berkeley, USA).
Results: Genetic analyses of the full length ORF26 show an overall variability of
2.1% (19 point mutations in a 915 bp region) among all analyzed HHV-8 variants,
with 78.9% of mutations (15 pm) clustered in a 167 bp fragment (nt 349-516)
where the variability peaks at 9.0%. These hypervariable region is characterized
by a low antigenic index and a prevalence of synonymous (S) versus non synonymous (N) mutations (ds/dN = 2.314). The genetic variability of the ORF26
allows the phylogenetic characterization of HHV-8 variants and the identification
of three major branches with bootstrap values >90. ORF72 is highly conserved
between representative members of each branch with homology >99%; on the
other hand the variability present in the K1 gene, particularly within repetitive
regions, hampers so far our phylogenetic analysis.
Conclusions: These data confirm our preliminary, results on the 233 bp fragment (nt 355-587) (Vancouver, 1996). Point mutations for all isolated HHV-8 are
clustered in the central region of the ORF, and allow by phylogenetic analysis the
identification of three major branches, reminiscent of the three strains identified
for mutation patterns by Zong et al., 1997. HHV-8 variants prevalently show an
ethnic/geographical distribution without pathogenic relevance; specific mutations,
however, were reported for lymphomas-related HHV-8 isolates (E.E. Kaaya et al.,
ECEAR 1997). Sequencing analysis data obtained on ORF72 and K1 are, so far,
not relevant for HHV-8 phylogenetic studies.
KS working group: A Favero, ML Calabro, L Chieco-Bianchi (Univ. of Padua, Italy);
M Cusini, L Brambilla, E Alessi, AF Finzi, (Univ. of Milan, Italy); S Gafa (Arcispedale
SMN, Reggio, Emilia, Italy); A Hatzakis (Univ. of Athens, Greece); R Downing,
B Biryahwaho, SDK Sempala (UVRI, Entebbe, Uganda); B Safai (MSKCC, NY,
USA); A Caterino-de- Araujo, VAF Alves (Inst. "Adolfo Lutz, Sao Paulo, Brazil).
Supported by Ministero Italiano della Sanita (Ric. Corrente 1998), X Prog.
AIDS-I.S.S (1998), and the ICSC-World Lab - Lausanne (Project MCD-2/7).
31194 Presence of HHV8 antibodies correlates with sexual
behavior in young homosexual men
Jay A. Levy1, D.J. Blackbourn1, E. Lennette2, D. Osmond1. 1UCSF, Dept.
Medicine, RM. S-1280 San Francisco, CA 94143-1270; 2 Virolab Berkeley CA,
USA
Objective: Determine the HHV8 serologic status in a cohort of young homosexual
men and evaluate the behavioral risk factors associated with HHV8 infection.
Methods: Subjects recruited from the San Francisco Young Men's Health Study

(SFYMHS) were evaluated annually for HIV infection and a complete questionnaire about their sexual behavior in the past year. Plasma samples were tested for
anti-HHV8 antibodies by an immunofluorescence assay. Using the HHV8-infected
lymphoma cell line, BCBL-1, two antigen staining patterns are evident: a latent
antigen present in both non-activated and activated cells and a lytic or replicative
antigen present only in activated cells.

Results: This point prevalence study was performed on two groups of SFYMHS
subjects consisting of 79 individuals. In the first group of 39 people, 4 were positive
for antibodies to the latent nuclear antigen associated with development of KS
and these 4 also had antibodies to the lytic antigen. Another 11 individuals were
positive for anti-lytic antigen antibodies only. No association of HHV8 seropositivity and HIV infection was noted; only 6/39 subjects were infected with HIV. 18%
of individuals reporting no receptive anal intercourse were HHV8 seropositive
compared with 70% who reported 10 or more receptive anal intercourse partners
in the last 12 months. Unprotected, receptive anal intercourse with more than
2 partners correlated with HHV8 infection. A second group of 40 subjects gave
similar results.
Conclusions: There is a strong association between infection with HHV8 and
sexual behavior, particularly receptive anal intercourse.
31195 Antibody response against HHV-8 by
immunofluorescence assay (IFA) using BC-3 and BCP-1
cell lines
Jean-Pierre Couty, Francois Denis, Sylvie Ranger-Rogez, Jean-Philippe Rogez,
Pierre Weinbreck. Deparment of virology Chru Dupuytren 2, Avenue Martin
Luther King 87042 Limoges cedex Limoges, Deparment of tropical and
Infectious Diseases Limogs Chru, Dupuytren, France
Objectives: To compare the two immunofluorescence assays using respectively
BC-3 and BCP-1 cell lines to detect antibodies directed against lytic and latent
HHV-8 antigens.
Design: Choose the more sensitive assay to detect antibodies present in sera
from HIV-infected patients presenting Kaposi's sarcoma (KS).
Material and Methods: Serum samples were obtained from 30 HIV-infected
patients presenting KS, one HIV-negative patient suffering from Multicentric
Castleman's Disease (MCD), 50 HIV-negative pregnant women and 50 healthy
blood donors. The two cell lines used (BC-3 and BCP-1) were induced by tetradecanoylphorbol acetate (TPA). Slides were saturated, then incubated with twofold
serial dilutions of sera and with fluorescein anti-human IgG F(ab')2 fragments.
Results: A nuclear immunostaining was visualized under non induced conditions (latent nuclear antigens). A cytoplasmic staining occurred when cells were
exposed to TPA (lytic antigens). Immunostaining was more intense with the BC-3
than with BCP-1, improving the visualization and allowing elevated antibody titers
detection. HHV-8 specific antibodies were detected in 83.33% serum samples
from AIDS-KS patients, 2% from pregnant women and 2% from blood donors.
Uninfected lymphoblastoid cell line (HSB-2) used as negative control remained
consistently negative. Anti-EBV antibodies detection was investigated for both
IgG anti-EBNA and anti-VCA, and there was no correlation between antibodies
directed to HHV-8 antigens and those detected against EBV. However, HHV-8
DNA sequences were detected in 4 out of 17 PBMC sample tested (obtained concurrently with the serum) among the AIDS-KS patients who presented elevated
HHV-8 antibody titers.
Nearly similar results concerning HHV-8 seroprevalence have been reported
by several authors. BC-3 cell line under induced conditions allows to obtained
elevated HHV-8 antibody titers and improves the microscopic visualization.
31196 Human herpesvirus 8 (HHV8) infection of peripheral
blood cells (PBMCs) in HIV+ women and HIV-controls at
risk: Women's Interagency HIV Study: WIHS
Alexandra M. Levine1'5, Antonella Maffei2, J. O'Leary2, S.L. Melnick3,. Silva2,
R. Greenblatt4, D.M. Knowles2. USC/Norris Cancer Hospital MS 44 1441
Eastlake Ave. Los Angeles, CA; 2New York Hospital - Cornell Med College, New
York, NY; 3National Cancer Institute, Bethesda; 4Univ. of California San
Francisco, San Francisco, CA; University of Southern Califonia/Medicine, Los
Angeles, CA, USA
Objectives: To determine the prevalence of HHV8 infection by polymerase chain
reaction (PCR) in PBMCs from HIV+ women and their FHV-controls enrolled in
the WIHS, and to correlate HHV8 infection with risk factors for HIV infection, and
with clinical illness.
Design: Prospective, longitudinal, multi-center study in 6 sites in the USA
Methods: Cases (adult HIV+ women) and controls are seen every 6 months.
PBMCs are stored, and an extensive questionnaire is administered. DNA extracted from stored PBMCs. PCR amplifications performed with KS330 primers
within ORF 26 of HHV8, using TaqMan PCR. Positive HHV8 required reproducible
amplification in 2 independent reactions. DNA from PBMCs of healthy males was
used as negative control on each run. 160 HIV+ and 42 HIV- women from LA &
SF were studied.
Results are presented in the table.
HIV+/HHV8+ HIV+/HHV8- HIV-/HHV8+ HIV-/HHV8 -Number (%) 29 (18%) 131 (82%) 6 (14%) 36 (86%)
Mean age 35.3 (22-52) 35.4 (19-66) 31.3 (20-41) 33.4 (22-53)
Median CD4 293 (0-1509) 325 (7-1102) 922 (537-1270) 1090 (547-1910)

HX IDU 5 (17.2%) 33(25.2%) 3 (50%) 14(39%)
Anal sex w male 10 (35%) 37 (28%) 1 (17%) 15 (42%)
Bisex male partner 8 (28%) 30 (23%) 0 7(19%)
Partner w/KS 0 0 0 1 (2.8%)
CDC class C 13(45%) 35 (27%) 0 3 (8%)
HX KS in patient 0 0 0 0
Hx any cancer 4 (14%) 15 (12%) 0 2 (5.6%)