Abstracts Vol. 1 [International Conference on AIDS (11th: 1996: Vancouver, Canada)]

Track A: Basic Science

Mo.A.1001 - Mo.A.1006

Mo.A.100 I1
THE DYNAMIC EXPRESSION OF CD4 ON CULTURED MONOCYTES:
IMPLICATIONS FOR IN VITRO HIV INFECTION
Graziani, Gina Maria, Filion, L.G. University of Ottawa; [Dept. of HL,. 1;,& Immunology,
Ottawa, ON, Canada
INTRODUCTION: Following overnight culture of purified mon, ' i rocyt:es in a
PBMC (peripheral blood mononuclear cell) fraction, CD4 is dow, ".L 1 Howeve, within the PBMC cultures, CD4 on T cells continues to be expressed, i. t CD4 regulation is cell-specific.Whereas much work has been done on T < I li > u1 ration and the
role of p56lck and serine residues within the cytoplasmic tail, littlc i Ik r, iBout the regulation of CD4 on monocytes.
Objectives: To determine the fate of monocyte CD4 following,)%,'i:!i,ie.,and to
determine the mechanisms responsible for the down-regulation.W V hypothesized that
monocyte CD4 is internalized following overnight culturing, and that this internalization is
triggered by the adherence of the monocytes to the culture sui te iis ' r.y undergo differentiation into culture-derived macrophages. Furthermore, we poi Isd it, it thie differential
regulation of CD4 on rmonocytes and T cells may be the result of difcrentiI signal transduction pathways, specifically at the level of the serine residues, whi s would be reflected by
differences in the transmembrane and/or cytoplasmic tail sequencs olf th molecule.
Methods & Results: Flow cytometric analysis revealed that the down-i egulation of monocyte CD4 is the result of internalization of the molecule (n - 10). Culturing PBMCs in
Teflon vials (which inhibit adherence) and in gelatin/fibronectin coated fLsLs suggests that
the down-regulation of rnonocyte CD4 is not mediated by adherence (r -). RT PCR and
automated sequencing of the CD4 molecule of uncultured, purified pieri pheral blood monocytes and a promonocyte cell line (U937s) reveals that their transnrembrane cd cytoplasmic domains are identical to that of the published T cell sequence.
Conclusions: We conclude that monocyte CD4 is internalized following overnight culturing.
However adherence of the monocytes to the culture surface does not appear to mediate
this internalization. Furthermore, the transmembrane and cytopiasrmic.rsequences of ronocyte and T cell CD4 are identical, suggesting that the differential expi rssioi of the molecule
by each cell type is not a consequence of differences at the level of the serienne residues.
These results may have important implications with respect to the isn vitro role of CD4 as
the major receptor for HIV in monocytes and mnacrophages.
Graziani, G. M. - University of Ottawa, Department of Microbiology/r risir nIoigy, Faculty of
Medicine, 451 Smyth Rd. Ottawa, ON, CA K I H 8M5 tel: [phone] ext 83 I; Fax: [phone]; e-mail: [email protected]
Mo.A. 1002
THE IMPLICATION OF CD26 IN HIV INFECTION:VIRAL ENTRY AND ITS
CYTOPATHIC EFFECT
Christian Callebaut, Etienne Jacotot, Bernard Krust, and Ara G. Hrvanessi,, iiinst itut Pasteur,
Unite VIC, UA CNRS II 57, Paris. France
Objective: TheT cell activation antigen CD26 is a cell surface senne exop iptids e charater
ized by a dipeptidyl peptidase IV (DPP IV) activity We have previously retoited that CD26
through a potential interaction with the V3 loop of HIV envelope gp I:?0I,, ould serve as a
cofactor of CD4 in entry of lymphotropic HIV I Lai and HIV-2 fH/ isoltesc. Recently the
role of CD26 along with the V3 loop was emphasized for entry ad i s,cytocpathic effect
using monotropic HIV I isolates (Oravecz et al., 1995; Nature Medi ne I, 9 19); and on the
other hand, a V3 loop peptide was shown to bind a synthetic ICD2 peptid e containing a
catalytic domain involved in the DPP IV activity (Chin et al., 1995; inrmmun. l tier 44, 25).
Methods: Because of the existence of species specific restrictions or IiV entr y and replication, the role of CD26 or any other cofactor has to be demonstrited in turani cells.
However, as almost all CD4+ cells permissive to HIV infection express l ow but reproducibly
detectable levels of CD26, we selected two such cells, CEM and Jurkt, ir order to establish
by transfection cell lines expressing enhanced levels of CD26.The entry w as monitored by
the detection of proviral DNA synthesis and by the kinetics of virus pi oduction, whereas
initiation of cell death by apoptosis was monitored by infection of the diffi rent cell lines
with a vaccinia virus recombinant expressing HIV gp I 20/gp4 I complex (VV env).
Results: The entry of HIV I Lai was found to be accelerated by 24-48 hr isix independent
clones of CEM cells expressing enhanced levels of CD26 compared with six other corresponding control clones. As a consequence of different kinetics of i rfe,:tion in low and high
CD26 expressing CEM cells, the occurrence of cell death was at lea ist two days earlier in
high CD26 expressing cells. In a separate set of experiment using Jutr kEit cell lines, we could
demonstrate that after I 6 hr of infection with VV env, the HIV gp 120/p4t iinduc ed apoptosis was observed only in cell lines expressing enhanced levels of CD26.
Conclusions: These results indicate that the level of CD26 could deternir- the rate of viral
entry and its cytopathic effect, 2 events which are initiated by the inter ctir of gp 120/gp4 I
complex with cell surface CD4.
C. Callebaut, Institut Pasteur, Unite VIC, UA CNRS 1157, Paris, Fr, nc' iel 33.1.568 8896
Fax 33 I 14061 3012 email: chrcall(epasteur.fr
Mo.A. 1003
IDENTIFICATION OF THE 02-m DERIVED EPITOPE RESPONSIBLE FOR
NEUTRALISATION OF HIV ISOLATES
Carole Le Contel, Pascale C alea. Franoise Silvy Ivmn Hirsch, Jean C de ( I an',n
INSERM U322, Unite de Recherches sur les Retrovirus et Maladie, Asso c, I'm
Scientifique et Technologique de Luminy BP 33, 13273 Marseille:edex 9 FRANCE.
Objective: Specific antibodies against 2-nicroglobulin (f2-m) have 1een deis nstrated to
precipitate HIV I LAV showing that HIV virions carried 532-m on ithei sriice. Such antibodies inhibit replication of HIV I LAV.We investigated whether $2 irs could represent a
target against all HIV strains and which 12-m epitope is responsible fIr neutralization.
Methods: Neutralization assays with anti-human [52-m monoclonal iit todies (anti hu-52-rn
MAbs) were performed with different HIV isolates on peripheral blood lmphocytes culture
(PBL).The study included laboratory adapted, Zarian virus HIV I N.)K, m aophage tropic
strain HIV I PAR and primary clinical isolates including newly identified siruses from the
International HIV I Isolates Panel. By using overlapping synthetic peptIde (1 derived
f6-om the amino acid sequence of the human 52-m and their derived shorter forms (7 aa)
we determine which epitope prevents neutralization induced by anti hu-i$2-m MAbs

through aT cell lne cytopathic assay and RT production assay on HIV infected PBL.
Results: Anti hu (32-m MAbs BI G6 and B2.62.2 inhibited at 70 95% the production of all
tested viruses ( 1 00 TCID50 dose) in primrary leukocytes.The cytopathic effect of HIV I
NDK on a Tcell line was inhibited by B I G6 MAb (5 rg) and this inhibition was reversed by
addition of four specific 14 as peptides (100 pg) derived from the hu [2 rm aa sequence.
We showed that 3 out of the 4 actives peptides shared a coem on amino acid motive
(PKI).Their shorter formns (7 aa) around the particular proline residu) were more efficiently
recognized by B IG6 MAb in an ELISA developped in our laboratory Theses 3 specific 7 aa
peptides (R/V, F7E, S7K) also prevented neutralization of HIV NDK induced by BIG6 MAtb
on PBL. Among them R7V, located at the N terminal part of the 32 tm, was found to be the
most efficient on the virus neutralization.
Conclusion:We selected a (32 -m derived peptide. R7V, which becomes a potential target
for vaccine agaiust HIV. Our results suggest that utilization of non-polymrorphic human cell
protein derived peptide present on all tested HIV isolates and eliciting universal HIV neu
tralizing antibodies may constitute a promising vaccine strategy
C Le Contel, INSERM Unite 322, B.P 33, 13273 Marseille Cedex 09, Fran.e.Telephone: (33)
91 82 1 75 0-Fax [phone]
Mo.A.1004
DIFFERENTIAL COURSE OF HIV-I INFECTION IN PRIMARY HUMAN MONOCYTES
AND MACROPHAGES
Schneider osef, Bohuschke M, Kary B, Herchenroder O. Department ofVirology, University
of Freiburg, Freiburg, Germany
Objective: Blood monocytes can transport HIV from the blood into the organs. As a pre
requisite to understand their potential role in the AIDS pathogenesis, we have studied the
course of HIIVinfection during differentiation of blood monocytes (MO) to adherent
macrophages (MF in vitro.
Methods: MO from 34 healthy blood donors were purified by ficoll gradient centrifugation,
countercurrent elutriation, and 2 hrs adherence.The purified cell populations consisted of
more than 98% MO, which differentiated to MF within 6 days of adherent culturing in
medium containing 10% of human serum. MO were infected with a low MOI of the
mracrophage-tropic HIV- I BaL strain at the day of isolation or six days later after differentia
tion to MF.Virus production was measured in the culture fluids by a commercial HIV p24
ELISA. Provirit DNA and Virus RNA were detected by nested PCR.
Results: All cultures infected as MF produced virus within 5 days. In contrast infection of
MO resulted in spontaneous virus production only in cultures fl-rom 34% of the donors. In
another 139% of infected MO cultures, virus production could be induced byTNF- and
GM -CSt MO cultures of 47% of the donors remained virus-negative despite this stimulation. Ful-length HIV DNA was detected in all cultures after the lourth day of infection,
whereas viral RNA was found only in the virus producing cultures.
Conclusions: Monocytes of all donors were infected. However, in fireshly isolated MO from
the majo rty of donors virus replication is inhibited at the level of transcription.This block is
not apparent in differentiated MF, and nriot regularly released during differentiation of MO to
MF. he observed variation of virus replication in monocytes of individual donors may contribute to understanding of the individual variations in HIV-induced pathogenesis and should
therefore be studied in more detail.
Dr Josef Schneider Abt.Virologie, Hermann-Herder Str: I I, D- 79104 Freiburg Tel.: *4976I
2036588 Fax: [phone] email: schfadsun I.ukl.uni-freiburg.de
Mo.A. 1005
SUBTYPE IDENTIFICATION AND REPLICATION CAPACITY OF HIV-I STRAINS
ISOLATED IN HUNGARY
NazyKaroly, Barabiss E.,Varkonyi V., Hoarvath A. National Institute of Dermato- Venereology
Budapest,-lurunary
Objective: To identify HIV I envelope sequence subtype and characterise in vitro replication
capacity of HIV strains from infected asymptomaratic individuals.
Methods: DNA samples derived from periferal blood mononuclear cells were analsed by
sequening of HIV- I env region encodingV3-loop. A 211 nucleotide fragment (7063-7274)
of I-ilV I nv, containing the entire V3 region and adjacent sequences were amplified by
PCR using appropriate oligonucleotide primers. Nucleotide sequence alignment companison
was made to the env region of HXB 2, HIV I BRU(LAV I) and IIV- I NL43(NYS).
Infectious HIVs were isolated by cocultivation and replcation capacity of the isolates were
determined in 5 permanent cell lines with lymphoid or macrophage orngin.
Results: The mmmv sequences derived from individuals infected in Hungary belong to subtype
B HIV I. figh similarity (96.9%) to env V3 region of HIV I BRU suggests strong subtype
association with IAV -I. Replication analysis shows the HIVs isolated are with the rapid/high
(PR/H or SI) phenotype.
Conclusions: t-he delayed occurence and still low rate of the AIDS epidemic in Hungary
raise the possibility of the circulation of HIV subtypes with altered replication capacity and
pathogenecity compared to those strains of HIV detected in Western Europe and the
ISA.The env basied classification and the biological characterisaton of HIV subtypes and
knowledge of their global incidence and prevalance help further understand geographical
distribution and functional significance of diversity in relation to transmission and pathogenesis f LI.
(Suppored pa rtlI by the Nation ml AIDS Cormittee under #4.1.95)
K.Nagy Maria St.4 I. Budapest, Hungary H1085tel:+36 I 210 0310 Fax f;6 I 134 0566
i-n l: nauqkai(i aborsote.hu
Mo.A. 1006
SEPARATION OF GPI20 AND GPI60 BY CIBACRON BLUE 3GA
Toshio Hattori i to Y*, Kubo T*, Zhan X*, Sakaida H, Nishikawa S**, Uchiyama T
tKyoto Univ. "Vakenyaku CO., LTD, Kyoto, Japan
Objectives: \Ne have attempted to separate gp 120 from gp I 60 firom the culture super
runts of gp120/gp 60 secreting CHO cells to characterize the biological and biochemical
features if both proteins.
Methods: CHO SEC cells were used for secreting cells (kindly provided from Dr:Weiss at
FDA).The cells are made by transfecting HIV-I (HXB-2) envelope gene (5999-8270).The
cells secrete transmembrane-portiorn-truncated gpl60 and gpI20 in culture medium. A

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