Final Program and Oral Abstracts [International Conference on AIDS (8th: 1992: Amsterdam, Netherlands)]

MoA 0048-MoA 0051

MONDAY, 20 JULY 1992

MoA 0048 RECEPTOR-MEDIATED ACTIVATION OF THE VIRAL ENVELOPE INVOLVES THE EXPOSURE OF
NOVEL NEUTRALIZING ENVELOPE DETERMINANTS. Allan. Jonathan S.: Whitehead, E.M.;
Strout, K.; Shuler, K.; Kanda, P.; Southwest Foundation for Biomedical Research, San
Antonio, TX, USA
Obiectivesa Interaction of simian immunodeficiency viruses from African green monkeys
(SIVagm) with soluble receptor molecules (sCD4) results in profound enhancement (100-fold) of in vitro
infection in contrast to sCD4 blocking of HIV-1 and HIV-2. We have previously shown that this enhancing effect
is a direct consequence of gpl 20 interaction with CD4 and that viral enhancement could be neutralized with
antibodies from infected green monkeys once virus has been treated with sCD4. This finding suggested that
sCD4 induces important conformational changes in the virion spike (activation) and that this transitional state
is part of the entry process. Mapping of neo-neutralizing domains on SIVagm may shed some light into the
overall mechanisms of viral entry for this family of immunodeficiency viruses and provide for novel
approaches in vaccine and therapeutic strategies.
Methods: To define neutralizing domains on SIVagm, monodonal antibodies were prepared by immunization of
mice with whole virus. Monoclonal antibodies with defined specificity were identified by screening in a
functional assay for neutralization of sCD4-mediated enhancement of SIVagm infection. In a second approach,
we synthesized synthetic peptides to defined epitopes of SIVagm gpl20 and gp36, prepared hyperimmune sera
from immunized rabbits, and antibodies were assayed for neutralization of sCD4-treated SlVagm
Resultse Monclonal antibodies were produced that functionally blocked sCD4 mediated enhancement of SIVagm
and mapped to gpl 20. By radioimmunoprecipitation analysis (RIPA), binding of one antibody (AG1.0) to gpl 20
was enhanced by prior incubation of virus with sCD4 indicating that this neutralizing determinant was further
exposed upon sCD4 activation. in addition, polyclonal antibodies to a gp1l20 synthetic peptide were also found to
neutralize this sCD4-mediated enhancement of SIVagm. Remarkably, monoclonal antibody (AG1.0) also bound to
this synthetic peptide providing convincing evidence for the identification of an immunodominant neutralizing
epitope involved either directly or indirectly in viral entry.
Conclusions: The study of receptor-mediated enhancement of SIVagm has allowed for the determination of novel
neutralizing epitope(s) that are exposed upon receptor-binding. Such novel domains may be involved in
secondary events in viral fusion and may prove useful in the design of more effective in vaccines and postinfection immunotherapeutic regimens.
Allan, Jonathan S., Dept. of Virology and Immunology, Southwest Foundation for
Biomedical Research, West Loop 410 at Military Dr. San Antonio, TX 78228, USA
Telephone: [phone], FAX [phone]
MoA 0050 GP120-INDUCED CONFORMATIONAL CHANGE IN CD4 IS IMPORTANT FOR
HIV-MEDIATED SYNCYTIUM FORMATION.
Manzoni,C. and Mous,Jan. Pharma Research New Technologies,
Hoffmann-La Roche Ltd,Basel, Switzerland.
OBJECTIVE: To study the mechanism of HIV-entry and identify novel ways of
interfering with post-binding steps.
RESULTS: During our mutational analysis of HIV- gpl20 - CD4 interaction, we identified a peculiar CD4 mutant, called Vl*, which was characterised by a different
folding pattern ( no binding of Leu3a or gpl20 ) and by the ability to enhance the
spread of HIV infection by promoting cell fusion.MAb raised against mutant CD4 Vl*
inhibited this enhancing effect, whilst e.g. Leu3a could not block the Vl*-induced
syncytium formation. Moreover, V1* mAb displayed HIV neutralising activity, although
it neither bound to native CD4 nor to gpl20.This surprising result indicated that
V1* mAb interfered with a step after binding of gpl20 to its receptor CD4.
Immunological binding studies indicated that after binding of gpl20 to CD4, the
receptor underwent a conformational change mimicking Vl*. This became evident by the
fact that Vl*mAb, which recognizes Vl* but not native Vl, interacted with CD4 only
when complexed with gpl20.Initial studies also indicated that recombinant V1* interacts with a specific receptor on T-cell lines. This finding suggests that the
binding of gpl20-induced Vl* like CD4 determinants to this factor X is important for
the induction of membrane fusion, which would explain both the fusion-enhancing
effect of recombinant Vl* and the neutralizing activity of anti-CD4 V1* mAb.
CONCLUSION: We identified a novel HIV neutralisation principle which is based on
the interference with a specific step after the binding of gpl20 to its receptor.
Mous, Jan, F.Hoffmann-La Roche Ltd, CH-4002 Basel, Switzerland.
Tel. [phone], Fax: [phone]
NOTES

MoA 0049 Claham. P: McKnight, A'; Simmons, G'; Cordell, J*; Dean, C'*; Weiss, R'.*
Chester Beatty Labs, Institute of Cancer Research, Fulham Road, London. SW3 6JB.
UK. *" Haddow Labs, Institute of Cancer Reseach, Sutton, Surrey S2 5NG. UK.
CD4 Independent Entry by HIV-2: Properties of an alternative cell surface receptor.
Introduction and obiectives;We have recently shown that a substrain of HIV-2ROD (LAV-2/B) efficiently
infected CD4-negative human RD, Daudi and Rai cell lines. Other HIV-2 strains either failed (e.g. CBL20) to infect or infected less efficiently (e.g. SBL6669) than LAV-2/B, although most HIV-2 isolates
tested induced syncytia if treated with soluble CD4. These results suggest that HIV-2 can use an
alternative cell surface receptor independently or in conjunction with CD4 (J. Virol. In press). Here we
studied the distribution and properties of this receptor on human cell types. We also present a
preliminary characterization of rat monoclonal antibodies raised against LAV-2ROD that inhibit CD4 -independent entry.
Methods: HIV-2 infectivity and cell to cel fusion assays in the presence and absence of inhibitory CD4
mAbs were used to assess expression of non-CD4 receptors on different cell types.
Results: The receptor for HIV-2 CD4 -independent entry was found to be expressed mainly on primary
and established haematapoietic cells. Several rat mAbs raised to LAV-2ROD recombinant gp105
specifically inhibited CD4-independent entry, but did not affect CD4-dependent entry, whether CD4
was present in a soluble form or expressed at the cell surface.
Conclusions: An altemative cell surface receptor that permits HIV-2 infection was found to be mainly
expressed on haematapoietic cell types. These results may have implications for the in vivo tropisms
of HIV-2
Paul R. Clapham, Chester Beatty Labs, Institute of Cancer Research, Fulham Road,
London. SW3 6JB. UK. Tel. [phone]. Fax [phone].
MoA 0051 HIV-1 VIRION-ASSOCIATED OUTER ENVELOPE
PROTEIN gp120 IS CLEAVED AT THE V3-LOOP BY INCUBATION WITH RECOMBINANT SOLUBLE (rs) CD4. Werner, Albrecht; Struble, H.K.; Levy,
J.A. Cancer Research Institute, Department of Medicine, University
of California, San Francisco, CA, U.S.A.
Objectives: To examine whether proteolytic cleavage of the HIV-1 V3 loop is involved in the virus
infection of peripheral blood mononuclear cells (PMC).
Methods: Molecularly cloned and biologically active HIV-1 strains were grown in human PMC. Titration and blocking experiments with rsCD4 were performed in PMC. Virus was purified by filtration, centrifugation through a 28% sucrose cushion, and by sucrose gradient banding. Further purification was
done by Sephacryl S-1000 chromatography. Virus suspensions were incubated with rsCD4 at 37' C to
determine sensitivity to this molecule. Virus proteins were visualized by immunoblot procedures.
Results: HIV-1 virus strains showed various sensitivities to the inhibition of infectivit b rsCD4
Virus strain TCIDO50 TCID50 with 10tOg/ml rsCD4 ATCID50
HIV-ISF33me 104.69 <100 >10469
HIV-ISFt3mc 105.7 102.78 102.92
HIV-ISF2mc 104.21 102.66 101.55
HIV-1SF162mc 105.16 104.69 100.47
In addition, incubation of purified virus suspensions with rsCD4 led to cleavage of virion-associated
gpl20 at the V3 loop in a time and rsCD4 concentration-dependent manner. Purified rgpl20 served as
the negative control, and is not cleaved, even when contaminated with a HIV-2 virus suspension, purified in the same manner as HIV-1. The cleavage can be partially inhibited by PMSF and completely by
incubation with a monoclonal antibody directed against the V3 loop. The cleavage time of nonrsCD4
inhibitory virus strains (e.g. HIV-1SF162) are at least two times longer than that for viruses that can be
blocked easily.
Conclusions: Exposure of different virus strains to rsCD4 shows varying sensitivity to reduction in
infectivity. Concomitantly, this incubation leads to cleavage of virion associated gp120 at the V3 loop in
a time and rsCD4 concentration dependent manner, the results suggest that virion associated gpl20 is
subject to cleavage after attachment with CD4.
Werner, Albrecht, Cancer Research Institute, University of California, San
Francisco, CA 94143-0128, USA
Tel: [phone] FAX: [phone]
NOTES

Mol4