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

Track A: Basic Science

Mo.A.1043 - Mo.A.1047

Methods: The F I12-HIV genome was inserted in the opposite orientation with respect to
the N2 retroviral vector, after replacing the nef gene with the hsumar inerve growth factor
receptor (NGFr) gene. CEMss cells were transduced with the N2iF l 2-HIV/NGFr retroviral
vector, selected by means of the NGFr marker, molecularly characterized and finally HIV- I
challenged. Moreover HIV-I chronically infected Hut-78 cells were ransd.ced with the
same retroviral vector. NGFr expressing cells were selected and (he HIV infectivity of the
supernatants was titrated as TCID50/ml on C8 I 66 cells.
Results: By transfecting HeLaCD4+ cells, we have demonstrated that the expression of the
non-producer F I2-HIV variant is able to induce resistance to HIV superinfection, through a
mechanism not involving CD4 expression (Virology 206: 76-84, 1995). By inserting the nef
deleted F I 2-HIV genome in the N2 retroviral vector, we recovered recombinant retroviral
pseudotypes able to transduce CD4+ human T cells (J.Virol. 69: 66 I8-6626, 1 995).We
demonstrated that F I2-HIV expressing neor transduced CEM s cell clones resist HIV superinfection at a m.o.i. up to 105TCID50/ 106 cells. In order to utilize the F 12-HIV interfering
properties in "ex vivo" cultures, we constructed the N2/FI2-HIV/NGFr retlroviral vector, so
that only those transduced cells exposing the truncated NGFr expressed the F I 2-HIV
genome.We demonstrated that uncloned CEMss cells transduced with the N2/F2 -HIV/NGFr vector and selected for NGFr expression resist HIV superir fection as do the
N2/FI 2-HIV transduced neor CEMss cell clones. Moreover, the infectivity of HIV released
f6om Hut-78 cells chronically infected by an HIV- I Italian isolate (I 0"TCID50/ml) was virtually abolished after transduction with the N2/F 1 2-HIV/NGFr vector and the selection of the
NGFr expressing cells.
Conclusions: We have demonstrated that the expression of the non-producer F 12-HIV- I
variant induces a block of the HIV life cycle both in acutely and chronically infected cells.
The recovery of N2/F 12-HIV/NGFr recombinant retrovirus will allow us to reproduce the
anti-HIV interfering phenomenon in "ex vivo" cell cultures.
M. Federico, Laboratory of Virology, Istituto Superiore di Sanite,Viale Regina Elena, 299
00161 Rome ITALY Telephone: [phone] Fax: [phone]
Mo.A. 1043
THE USE OF HIVVECTORS FOR AIDS GENE THERAPY
Matsukura Makoto*, Suzuki,T**, Shimada,T.**. * Dept. of Child Devel., Kumamoto Univ.
School of Medicine, ** Dept. of Biochem. & Mol. Biol., Nippon Medical School
Objective: To examine the feasibility of the use of HIV vectors in AIDS gene therapy.
Methods:The herpes simplex virus thymidine kinase (HSV-TK) suicide gene was inserted
between the packaging signal and the neo gene of pHXN, yielding pHXTKN.Te HSVTK
gene is directed by the HIV-LTR, while the neo gene is by the internal tk promoter.c Other
HIV vector plasmids are splicing mutant(pHXCmTKN), CMV pronmoter+TK (pHXCTKN)
and its reversed (pHXNCTK).
Results: CD4+HeLa cells and H9 cells were tranduced with the vectors arid were selected
with G4I8.The bulk cultures carrying HXTKN were found to be highly sensitive to GCV
one to two weeks after de novo infection of HlVlllb.The therapeutic index (ratio of GCV
IC50 between HIV uninfected cells and infected cells) was approximately two degree of
magnitude in MTT assay suggesting that HIV infection efficiently induced expression of the
HSVTK gene.While other cultures carrying HXN, HXCmTKN or HXNCTK are not sensitive to GCV, bulk culture carrying HXCTKN was profoundly sensitive to GCV even without
HIV infection.
Conclusions: T cell specific gene transfer, HIV specific gene expression and in vitro and in
vivo selection of HIV uninfected cells using the HIV vector may provide a novel strategy of
gene therapy for AIDS.
Makoto Matsukura, I - I - I Honjo, Kumamoto City 860, Japan Telephonc:96-373-5 197
Fax:[phone]
Mo.A. 1044
HIV-REGULATED LUCIFERASE AND DIPHTHERIA TOXIN A FRAGMENT GENES ARE
EXPRESSED SPECIFICALLY IN HeLa CELLS AFTER TRANSFECTION BY CATIONIC
LIPOSOMES
Konopka, Krystyna*, Harrison G**, SlepushkinV*, Feigner P***, Duzgbnes N.*. *University
of the Pacific, School of Dentistry, San Francisco, CA; **University of Colorado Health
Sciences Center, Denver, CO; *** Vical, Inc., San Diego, CA, USA
Objective: To determine whether HIV-regulated luciferase and diphtheria toxin A fragment
(DT-A) genes are expressed in HeLa cells following transfection by cationic liposomes and
whether expression of the DT-A gene in HeLa-T4 cells can protect against de rove HIV
infection.
Methods: HeLa/LAV and HeLa cells were transfected with pLUCA43 or pRSVLUC plasmids that encode the luciferase reporter gene under control of HIV- I or Rous sarcoma
virus promoters, respectively, complexed with LipofectAMINETM (LFA: DOSPA/DOPE
(3:1)). HeLaILAV and HeLa cells were transfected with pTHA43, an HIV-regulated DT-A
gene (HIV-DT-A), pTHA44 (a DT-A frameshift mutant of pTHA43) or pRSVLUC plasmids
complexed with LFA. HeLa cells were co-transfected with the HIV proviral clone, HXBABgl,
and pTHA43, pTHA44 or pRSVLUC plasmids complexed with I,2-dimyrstyloxypropyl-3 -dimethyl-hydroxyethyl ammonium bromide (UMRIE:DOPE (1:t)), HeLa-T4 cells transfected
with pTHA43 or pTHA44 complexed with UMRIE/DOPE, were infected with HIV-lIllIB.
Results: The HIV-regulated luciferann gene was expressed at a I 03-hold higher level in
chronically infected HeLa/LAV cells compaied to uninfected HeLas cells, while the extent of
expression of RSV-iegulated luciferase was the same in both cell lines. T-he delivery of HIVUT-A to HeLa/LAV cells did not have a specific effect on virus productiori in these cells,
since treatment of cells with control plasmids (pTHA44 and pRSVLUC) also reduced virus
production. Examination of the cytotoxicity of the added reagents indicated that the
observed effects were due to toxicity. Co-transfection of HeLa cells with boshl HIV-UT-A
and an HIV inohecular clone inhibited completely virus production. However, transfection of
HIV-U- A into HeLa-T4 cells before infection did not reduce p24 p-cr(ductios c:ompared to
the control plasmid (pTHA44).

Conclusions: HIV-regulated luciferase and DT-A genes were expressed specifically in HIVinfected or HIV-transfected HeLa cells following transfection by cationic liposomes.The
absence of protection against de novo HIV infection is most likely due to the DT-A gene not
being expressed in a sufficient number of cells.
K. Konopka, 2155 Webster Street, San Francisco, CA 94 115 USA Telephone [phone] I
Fax: [phone]
Mo.A. 1045
ENGINEERING T CELLS AGAINST HIV WITH HUMAN ScFvs SELECTED FROM
PHAGE-ANTIBODY LIBRARIES
Bitton Natacha*, PARIZOT C* SCHINDLER D,WAKST, DEBRE P* ESHHAR Z*,
GOROCHOV G* *lmmunologie, Piti-Salptrire, Paris, France and *Department of
Chemical Immunology, Weizmann Institute, Israel.
Objective: Engineering ofT cells that express A): chimeric receptors with an antibody-type
anti-HIV specificity that could eliminate infected cells in a non-MHc dependent fashion, or
B): intracytoplasmic antibody fragments directed against the HIV-h integrase.
Methods: Human single chain Fvs (ScFvs) are selected f6om: random combinatorial naive
antibody libraries or disease" libraries constructed with genes originating from in vitro
propagated and EBV transformed HIV patients B cells. ScFvs are then expressed without
leader sequence for intra-cellular immunisation or linked to the y chain of the FcyRIII for
surface expression in T cell lines.
Results: We have obtained several anti-ENV and anti-HIV-I integrase antibodies under the
ScFv format. Some of the anti-ENV ScFvs are able to bind the crude virus, and it is possible
to select phage-displ.iyed ScFvs using crude virus in order to obtain antibodies that bind
oligomeric gp I20. Chimeric receptors were then constructed under various formats with
or without spacer regions (Ig Hinge, CD4 Ig domains, CD8 Hinge, Myc Tag) and compared
in terms of optimal surface expression and antigen-induced cellular activation. In parallel, in
vitro affinity maturation of the antibody fragment was obtained using either a chain shuffling
approach or mutator strains.
Conclusion: Phage display of antibody fragments appear to be particularly well suited for
the selection and subsequent maturation of genes to be used in immunotherapy protocols.
N.BITTON, Immunologie Cellulaire, CERVI, CNRS URA625, Pitin-Salpmtri6re, 83 Bd de
l'H6pital, 75013 Paris, FranceTel: (33 I) 42 17 75 07. Fax: (33 I) 42 17 74 90,
email: gorichov~ccrjussieu.fr
Mo.A. 1046
ANTITAT GENE IS A CANDIDATE FOR BOTH T-CELLAND STEM-CELL GENE
THERAPY
Lisziewicz, Juianna *, Johnson, P**, Rosenzweig, M.**, Sun, D.***, Lorin, F.*. *Research
Institute for Genetic and Human Therapy Policlinico S. Matteo, Pavia, Italy, & Gaithersburg
(MD), USA; **New England Regional Primate Research Center, Southborough (MA), USA
***Laboratory of Tumor Cell Biology, NCI, Bethesda (MD) USA;
Objective: To determine the antiviral efficacy of the ntitat gene in in vivo infected T-cells as
well as in lymphocytes and macrophages differentiating from ontitat-transduced stem-cells.
Methods: Antitat is an autoregulated, dual function antiviral gene (polymeric-TAR and antisense-Tat comRbination) capable of inhibiting both HIV and SIV Retrovirus vector supernatant were used to transduce PBMCs isolated from AIDS patients or CD34+ stem-cells
fl-om normal macaques.Transduced BPMCs were cultured up to two weeks under conditions optimal for induction of HIV replication (PHA + IL2).We developed a technology to
differentiate entietat-containing bone marrow stem-cells to mature polyclonal lymphocytes
and macrophages. Cultures were expanded and analyzed for susceptibility to SIV infection.
Results: In cells isolated from AIDS patients entitat-gene inhibited the replication and the
cytophatic effect of HIV and allowed CD4+ cells to proliferate.pWe were able to demonstrate differentiation of transduced stem cells not only to macreophages but also to mature
lymphocytes. Mature, immunologically competent ontitat-containing lymphocytes were highly
resistant to SIV infection. Similarly, ontitst-containing macrophages were resistant to a challenge with a macrophage-tropic SIV. No toxicity was observed.
Conclusions: (i) Antitot is an effective antiviral agent in in vivo infected T-cells; (ii) Antitat
inserted into stem-cells can protect mature lymphocytes derived from these cells; to our
knowledge, this is the first demonstration that stable introduction of an antiviral gene into
hematopoietic stem-cells protected multiple liniages from viral replication; (iii) inhibition of
SIV with ontitot suggests that the monkey model can be used to evaluate both T-cell and
stem-cell AIDS gene therapy.
Julianna Lisziewicz, Research Institute for Genetic and Human Therapy, 7965 Cessna Ave.
Gaithersburg MD 20879, USATeI: [phone] Fax: [phone]
email: [email protected]
Mo.A. 1047
"PASSIVE" INTRACELLULAR IMMUNIZATION WITH AN EXONUCLEASE SPECIFIC
FOR SINGLE STRANDED DNA.
Savarno A,]urco F, Sinicco A, Pugliese A. University of Turin, Italy
Objective: A new molecule for experimental gene therapy was sought. We hypothesized
that an exonuclease specific for single-stranded DNA may inhibit HIV- I, since a single
stranded LDNA terminus (SSUT) extends from proviral DNA during its synthesis. An activity
independent 6-em a peculiar viral nucleotidic sequence was required. In fact, ribozymes and
annisense ohigonucleotides do not theoretically warrant a persistent activity, since a single
rnutation it HIV DNA may be sufficient to produce resistance.
Methods: UNAases were analyzed on the basis of the data presented in literature. MT-4
cells were infected with HIV- I 1118 at a MOI---100:1.The enzyme chosen was introduced
into the cells by means of a 3'incubation with a tensoactve solution.
Results: Enchericho coli Exo VII resulted to be the ideal agent for our purpose, since it
hydi oyzec SSUT in an absolutely specific manner and independently flutrn the nucleotidic
sequence. INo toxic effects were detectable in mock infected controls at the doses utilized,
while the intracellular expression of p24 resulted to be inhibited in a dose-dependent manrnecThe same solution without Exo VII did not interfere with viral replicaton. Both the presenice and the dose of Exo VII significantly compromised viral replication (p<0.001 ITwo-way
Ansova).

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