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

12th World AIDS Conference

Abstracts 41233-41236 789

least a 10-fold reduction in number of infected cells compared to HU/HU grafts,
and did not show obvious thymocyte depletion.
Conclusion: Our study shows that xenogeneic thymic transplantation may
permit the achievement of tolerance with restoration of immunocompetence in
humans using porcine tissues, which appear to be resistant to HIV. Thus, xenogeneic thymic transplantation has the potential to provide an important adjunct to
the therapy of HIV-infected patients.
41233 Characterization of T cell expressed chimeric receptors
with antibody-type specificity for the CD4 binding site of
HIV-1 gp120
Natacha Bitton, P. Debre, G. Gorochov. Cervi-Ura 625 - Hopital Pitie-Salpetriere
83, Bvd de I'H6pital - 75013 Paris, France
Objective: Engineering T cells that express chimeric receptors with an antibody type anti-HIV specificity that could eliminate infected cells in a non MHC
dependent fashion.
Methods: The human single chain antibody fragment (ScFv-B12) derives from
the IgG1B12 antibody1 directed to the CD4-binding site of gp120, a potent neutralizer of different HIV strains including a large panel of primary isolates. An scFv
fragment bearing the Pro- > Glu mutation in the complementary determining
region 3 (CDR3) of the heavy chain that improves 54-fold IgG1B12 affinity, called
scFvB12E, was also constructed. The scFvs were then expressed in the murine
MD45 T cell line under various formats, linked to the y chain of the FcyRIII with
or without spacer region (CD4 Ig-like domains D3 and D4 or CD8 hinge domain)
between the antibody domain and the signal transducing y chain.
Results: We tested the ability of the various transfectants to produce IL-2 in
response to gp120 stimulation. The different chimeric receptor formats behave
similarly in terms of scFv surface expression, but also according to their activation
threshold. We showed that cTCR engagement is not strain-specific since the T cell
transfectants could be stimulated with immobilized gp120 derived from various
HIV-strains. The native B12 antibody is able to bind to envelope proteins derived
from a large panel of primary isolates of HIV-1. To test the specific activation of
our transfectants against cells expressing envelop protein from such isolates, we
used BHK cells infected with Semliki Forrest virus-ENV (SFV-ENV) recombinants2
allowing expression of either laboratory HIV-1 strains-derived gp160 such as LAI,
MN, HXB2, MN12 or primary strains-derived gp160 such as BX08, CHARB or
LYON. Similar levels of IL-2 were secreted after co-incubation with cells expressing gp160 derived from LAI, BX08, HXB2 or MN12. The response of the
transfectants to stimulation with CHARB and LYON env-expressing cells was
weaker, but very significant. The latter result is contrasting with the lack of specific
staining of SFV-env (CHARB or LYON) infected cells by the IgG1B12 antibody, as
measured by cytofluorometric analysis. We also verified that HELA cells (which
constituvely express the coreceptor CXCR4) are able to bind HIV-1 gp160 when
transfected with the chimeric receptor scFv-B12-y, but do not become infected
by the virus.
Conclusion: Some primary strains of HIV-1 are resistant to neutralization by
IgG1B12. Indeed, no specific binding of IgG1B12 could be detected on target cells
expressing the envelope protein of some of the strains we tested. Nevertheless,
those infected cells were recognized by T cells with B12-like specificity. Our results suggests that the combination of T cell and antibody-based immunotherapy
could prove to be advantageous, particularly in order to target infected cells and
to inhibit cell to cell transmission of the virus.
[1] Burton D.R et al. Science. 266, 1024 (1994)
[2] Verrier FC. et al. Proc. Natl. Acad. Sci. USA, 94, 9326 (1997)
S412341 CD38 stimulation exerts antiviral effects and modulates
CXCR-4 expression in lymphoid cells
Liliana Calosso1, A. Savarino2, A. Pugliese2, F. Malavasi3. 'Via Santena 19
1-10126 Torino, Lab of Cell Biology of Genetics; 2University of Torino, Torino;
Background: CD38 is an activation marker of lymphocytes with co-stimulatory
and adhesion features. Our aim was to evaluate the role of CD38 in HIV-1
replication and its relation with fusin (CXCR-4) expression.
Design: In vitro study.
Methods: SUPT-1 cells or peripheral blood lymphocytes (PBLs) from healthy
donors were infected with the lymphotrophic strain HTLV-IIIB and co-cultured with
endothelium or monoclonal antibodies (mAbs) to CD38, CD28, CD26, CD4, gpl20
or CD19. Cell proliferation was measured by the MTT method. p24 in culture
supernatants was determined with an immunoenzymatic assay. Intra-cytoplasmic
p24 and CXCR-4 were evaluated by FACS analysis. The data were analyzed with
ANOVA test followed by Bonferroni's T test.
Results: HIV-1-infected SUPT-1 cells co-cultured with endothelium showed the
same proliferation rate as uninfected controls and this feature was partially abolished by mAbs that blocked the interaction of CD38 with its ligand. Cross-linking
of CD38 by mAbs mimicked the ligand function and prevented the decreased

proliferation of infected cells. To evaluate the effects of CD38 stimulation on HIV-1
replication, we stimulated cells with mAbs for different periods (from 24 hours
before to 48 hours after infection) and evaluated p24 production. In the presence
of anti-CD38 mAb, p24 was significantly lower than in controls (30% inhibition
even where the stimulation was begun 48 hours after infection). Since the spread
of HIV-1 among lymphoid cells depends on the presence of co-receptors, we
evaluated the effect of CD38 stimulation on CXCR-4. After 24 hours, CXCR-4

expression on SUPT-1 cells was unaffected, whereas it was down-regulated by
33% on PBLs cultured with IL-2 and anti-CD38.
Conclusions: Triggering of CD38 molecule in HIV-1 infected SUPT-1 cells
results in cell proliferation, cytophatic effects prevention and viral replication inhibition via a CXCR-4-independent pathway. However, CXCR-4 down-modulation
occurs in PBL after CD38 stimulation, suggesting a further potentially protective
activity against HIV-1 infection in human lymphocytes. These data may support
the use of CD38 stimulation in immunotherapy for HIV infection.
141235 Interleukin-2 (IL-2) treatment reduces soluble CD30
(sCD30) plasma levels and the percentage of apoptotic
cells in HIV infected patients
Cecilia Simonelli', S. Zanussi2, M. Dandrea2, M.T. Bartolin2, U. Tirelli', P. De
Paoli2. 'Division Medical Oncology and AIDS-CRO Via Pedemontana 12-33081
Aviano (PN); 2 Microbiology Unit-CRO, Aviano (PN), Italy
Objective: To determine the plasma levels of the soluble form of CD30 (sCD30)
and the percentage of apoptosis in peripheral blood mononuclear cells in HIV+
patients treated with 6 cycles (24 weeks) of antiretrovirals (AZT and DDI) plus
Interleukin 2 (IL2) in comparison to patients treated with antiretrovirals alone.
Methods: sCD30 values were determined by an ELISA assay on plasma samples collected before therapy (t = 0), after 2 weeks (t = 2), after 4 weeks (t = 4)
and after 24 weeks (t = 24) in 10 HIV+ patients with CD4 counts >200 <500/mmc
stage A2 B2 treated with a combination of antiretrovirals (AZT + DDI) and of IL2.
The percentage of apoptotic cells in the peripheral blood mononuclear cells was
measured at t = 0 and t = 24 by the cytofluorografic determination of DNA breaks
("TUNEL" assay). The controls were 10 HIV+ patients treated with antiretrovirals
alone.
Results: IL2 administration induces a rapid increase of sCD30 plasma levels
(167 1 128 U/ml at week 2, p = 0.02 compared to week 0). On the contrary, mean
sCD30 plasma levels are reduced in IL2 treated patients compared to patients
treated with antiretrovirals alone at the end of the treatment period (27.6 ~ 21
U/ml vs 59.7 ~ 22 U/ml p = 0.008). Finally, the percentage of peripheral blood
mononuclear cells undergoing apoptosis at the end of therapy is significantly
reduced in IL2 treated compared to antiretroviral treated patients (3.9 ~ 1% vs
9.5 + 2%, p = 0.009).
Conclusions: Our results demonstrate that a reduced immune activation is
achieved in IL2 treated HIV+. We therefore suggest that the beneficial effects of
this therapeutic protocol may depend not only on the capacity of IL2 to reconstitute a functional CD4 lymphocyte population, but also on its ability to reduce
immune activation: ultimately this could produce beneficial effects on HIV disease
progression..41236 HIV-1 polymerase chain reaction does not always
correlate with infectious virus in patients on passive
immunotherapy
Abraham Karpas', S. Ash2, D.R. Bainbridge3. iHaematology Department, MRC
Centre, Cambridge University, Hills Road Cambs; 2Ealing Hospital, London;
3Royal London Hospital, London, UK
Background: Polymerase chain reaction (PCR) is undoubtedly the most sensitive
assay for detection of HIV. In addition, there is a good correlation between HIV
disease progression decline in CD4+ T-cell count and increase in viral load as
monitored by PCR. However, in the case of patients who have been receiving
regular monthly transfusions of 500 ml of plasma from healthy HIV-1 infected
individuals containing a high level of antiviral neutralizing antibodies, the PCR
assay may give misleading results.
Methods: Plasma from AIDS patients before and after the infusion of plasma
with high levels of neutralizing antibody were tested for HIV-1 using three methods:
Viral isolation, Elisa for p24 antigen and PCR. Likewise, healthy HIV-1 infected
plasma donors were also tested with those methods.
Table. It shows that positive reactions in PCR for HIV-1 plasma RNA can be
recorded in some AIDS patients treated by passive immunotherapy (PIT) while
the plasma becomes negative for the viral p24 antigen in the Elisa test and fails
to yield infectious virus, indicating that the neutralizing antibodies killed cell-free
virus. Likewise, in some healthy long-term survivors, PCR positivity has been
recorded in spite of the fact that no infectious HIV-1 was isolated and the plasma
was negative for the viral p24 antigen.

HIV-1 HIV-1
isolation p24 antigen

PCR

AIDS
AIDS and PIT
Healthy HIV-1/long-term survivors

Conclusions: The results of our studies suggests that PCR which detects the
viral nucleic acid cannot distinguish between RNA of live or dead HIV. Therefore,
in the case of patients who receive PIT, the assay for the viral p24 antigen is likely
to be a more reliable reflection of HIV-1 neutralization.