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

Monday, July 8, 1996

Mo.A.406 - Mo.A.510

Mo.A.406
INDUCTION OF PRIMARY ISOLATE-NEUTRALIZING ANTIBODIES BY CANDIDATE
HIV- I VACCINES
Zolla-Pazner, Susan*, Berman P I, Gregory TI, Robert-Guroff M2, Naltuk RI, Sinangil F4,
Steimer K4. the NIAID AIDS Vaccine Evaluation Group. *VA Hosp. + NYU Med. Ctr, NY,
NY; I Genentech Corp., So. San Francisco, CA; 2Natl. Cancer Inst., Bethesda, MD; 3WyethLederle Vaccines, Pearl River, NY; 4Chiron Corp., Emeryville, CA USA
Objective: To determine if antibodies (Abs) that neutralize HIV i primary isoiates are
induced in chimpanzees and humans immunized with various candidate HIV- I vaccines.
Methods: Coded panels of sera from HIV-infected humans and from niri nfected humans
and chimpanzees immunized with various HIV- I vaccines were tested tor their ability to
neutralize low-passage Clade B HIV- I primary isolates using (a) a conventional neutralizing
assay with PHA-stimulated PBMCs as target cells, and (b) a new "resting cell assay" with
unstimulated PBMCs as target cells.
Results: (a) Sera of HIV-infected humans were able to neutralize both primary isolates and
lab strains of HIV (b) The sensitivity with which a virus can be neutralized nrd the level of
neutralizing activity in a serum preparation is determined by the conditions of the assay
used.The target cell used, as well as the particular virus and serumn specimen, each plays a
role in determining the sensitivity with which neutralization is detected. (c) The sera of
recipients of some vaccines were shown to contain primary isolate- neutralizing Abs if the
resting cell assay was used in which unstimulated PBMCs (rather than rritogen-stimulated
or transformed cells) were used as target cells. (d) In a chimp model, a correlation was
found between the presence of neutralizing Abs for laboratory and primary isolates and in
vivo protection.
Conclusions: These studies demonstrate that Abs can readily neutraize most, if not all,
Clade B primary isolates of HIV- I, that primary isolate-neutralizing Abs ar be induced by
some prototype HIV- I vaccines in humans and chimpanzees, and that the presence of neutralizing Abs in chimpanzees is a correlate of immune protection against HIV infection subsequent to an I.V challenge with cell-free virus.
Susan B. Zolla-Pazner, VA. Hospital, 423 East 23rd St., Room 18 I24N, NewYork, NY 10010 -5050 USATel: 212-263-6769 Fax: [phone] e-mail: ZOLLASe lo(MCRCR6.MED.NYU.EDU
Mo.A.500
STIMULATION OF HIV-I LTR-DIRECTED TRANSCRIPTION BY THE RAS PATHWAY
REQUIRES THE NOVEL TRANSCRIPTION FACTORS RBF-I AND RBF-2
BII, Sadowski, I. University of British Columbia,Vancouver, B.C., Cianada
Objective: We sought to determine the cis-acting DNA sequences necessary for the
stimulation of HIV- I transcription in response to the Ras signal transduction pathway in
JurkatT cells.
Methods: We used cotransfection of activated Ras alleles with HIV-LTR CAT reporter contructs to map the cis-acting DNA sequences essential for Ras responsive HIV transcription.
We have also employed electrophoretic mobility shift assays to identify novel DNA binding
proteins that interact with these Ras responsive sequences. Southwestern blotting was used
to determine the molecular weight of these DNA binding proteins. Finally site-directed
mutagenesis was used to show that binding of these factors to the HIV I LTR is necessary
for Ras responsive transcription in T cells.
Results: Ras responsiveness of the HIV- I LTR was found to reside 5' of the NF-KB sites as
well as immediately 3' of the TA-A box.These sequences bound specitically to nuclear factors from Jurkat cells.We have termed these factors RBF- I and RBF-2 for Ras responsive
element binding factors I and 2. RBF- I and RBF-2 have different DNA binding specificities
but both have DNA binding subunits of relative molecular weight 100 kDa. RBF -I binds to
an Ets like binding site but is distinct from Ets- I or Elf- I. RBF-2 binds near the LEF site but is
unrelated to LEF in DNA binding specificity molecular weight and reactivity with LEF antibodies. Site directed mutagenesis of the binding sites for RBF- I and RBF-2 demonstrated
that these factors are essential for Ras responsive HIV transcription.
Conclusion: The Ras signal transduction pathway impinges on the HIV- I LTR to stimulate
transcription.Two new DNA binding factors, RBF- I and RBF-2, are required for the
response of the HIV-I LTR to Ras.Thus, RBF- I and RBF-2 play a role in the induction of
HIV gene expression in T cells when the Ras pathway is activated.
Brendan Bell and Ivan Sadowski, Department of Biochemistry and Molecular Biology UBC,
2146 Health Sciences Mall,Vancouver; B.C., CanadaV6T IZ3.Tel. [phone]
Fax: [phone], e-mail: [email protected]
Mo.A.501I
REGULATION OF HIV TRANSCRIPTION IN T CELLS AND MACROPHAGES:
CRITICAL ROLE OF NF-KB ACTIVATION,AND ITS CONTROL BY NUCLEAR IKBs.
Virelizien Jean-Louis, Alcami, J. *Arenzana-Seis dedos, F, Italy R.T** Concer ted Action
("R'ocio" project) of Eu Biomed programme, and Unite dImmunologie Virale. lnstitut Pasteur,
Paris, France. *fHosp. 12 de Octubre, madrid, Spain. University of St. Andrews, Scotland.
Objective: How HIV uses the environment ofT cells and macrophages to benefit its own
latency and replication needs to be uinderstood to better- adapt future antnvwial inter-ventions to the original strategy of this human lentivirus.
Project: A network of european laboratories (project ROCIO, Concerted Action of the
BIOMED programme of EU) associated their complementary expertises to analyse the
molecular events controlling HIV genome transcription through occupancy of the HIV
enhancer by NF-KB in the two target cells of the virus:T lymphocytes and macrophages.
Results: In resting CD4 T lymphocytes isolated form the peripheral blood of normal
donors, the activity of the HIV LTR was found to be very low or undetectable.This transcriptionnal latency was correlated with the absence of active NF- Kb complexes in the
nucleus, and ceased upon T cell activation, when the viral enhancer sequence was occupied
by NF-KB. Such occupancy both initiated transcription and permitted HIV fat dependent
transactivation, which were both abolished in enhancer-mutated LTR vectors. A HIV
provirus with mutations in NF-KB responsive element could not est,:bhsh a productive
infection in peripheral CD4 cells.
In contrast to this strategy of latency/reactivation in T cells, HIV replicatioin in macrophages
was found to be self-perpetuating, through permanent NF-KB actaticon induced by viral
replication itself Blockade of this amplification loop by peptides inhibiting she pr cteasome
mediated IKB degradation aborted the permanent viral transcription observed in chronically infected U937 monocytic cells. Our enhancer-mutated HIV provirus failed to establish
productive infection in U937 cells.
We found that phosphorylation of the IKBa inhibitor leads to ubiquitination of the molecule, at lysine residues 21 and 22, thus dissociating NF-from IKBo. IKBce transcription
induced by NF-KB activation results in IKBo translocation into the nucleus, where the
inhibitor associates with NF-KB heterodimers bound to the HIV enhancer and terminates
its function.Thus occupancy of the HIV enhancer by NF-KB is sequentially controlled in a
positive and a negative way by post-translational events occuring at the level of IKBt upon
cell activation.
Mo.A.502
CLONING OF A NOVEL HUMAN PROTEIN THAT REGULATES NF-kB ACTIVITY AND
DELINEATION OF PHOSPHORYLATION EVENTS ON SPI: IMPLICATIONS FOR HIV-I
Jin DY Chun RF, Jang Kuan-Teb. Molecular Virology Section, LMM, NIAID, NIH, Bethesda,
MD, USA
Objective: To characterize the direct effector mechanisms that activate HIV- I LTR transcription factors Sp I and NF-kB by identifying novel regulatory second messengers and
phosphorylation events.
Methods: cDNA libraries were screeened to identify a novel human antioxidant (AOE372).
Full-length sequence of this cDNA was determined by dideoxy chain termination sequencing.The functional role of this cDNA was determined by co-expression with infectious HIVI molecular clones. Experiments assessing the role of phosphorylation were performed
using direct 32P-labeling of cells and in cell free extracts.
Results: NF-kB and Sp I are critical factors that regulate expression of the HIV- I LTR.
Although much has been described about NF-kB and SpI interactions with their cognate
DNA motifs in the LTR, little is understood about the direct molecular events, independent
of DNA-binding, that results in activation of these factors. We have noted that a second
messengers that control NF-kB activities inside cells is H202.We now report on the identification and cloning of a novel human antioxidant (AOE 372) that regulates the NF-kB activation of the HIV- I LTR. AOE372 is a pioneer cDNA; and we have deposited the full-length
sequence of this clone into GenBank (accession # U25182).We show that transfection of
AOE372 into cells regulates NF-kB activity and influences the expression from an infectious
HIV- I molecular clone. In complementary experiments, we have found that phosphorylation
at serine and threoine residues is a downstream activation event for Sp I. We demonstrate
that this event is mediated through protein kinase, DNA-PK and is modulated by the presence of the HIV- I regulatory protein,Tat. Cells deficient for DNA-PK activity were poor in
basal and Tat-activated LTR transcription.
Conclusion: Ouar recent findings have delineated two downstream events that activate transcription factors NF-kB and Sp I.These findings may allow us to better understand how to
modulate the expression of HIV- I in various cellular backgrounds.
Kuan Teh Jeang, Bldg. 4 Room 306, 9000 Rockville Pike, Bethesda, Maryland, USA
Tel: [phone] Fax: [phone] e:[email protected]
Mo.A.503
TRANSDOMINANT MUTANTS OF IKBa INTERFERE WITH HIV-I GENE
EXPRESSION AND REPLICATION
Hiscott *, Lin R*, Beauparlant P*, Kwon H*, Clarke M*, Gessani S**, Belardelli F**,
Wainberg M*. *Lady Davis Institute, McGill University Montreal, Canada; **lstituto
Superiore di Sanita, Rome, Italy
Objectives: NF-KB/Rel transcription factors participate in the coordinate activation of HIV- I
and cytokine gene expression. In the present study transdominant negative mutants of IKBa
were examined for their ability to interfere with Tat-TNF activation of the HIV- I LTR and
HIV I1 replication.
Methods: Point mutations of IKBo were generated by overlap PCR mutagenesis using Pfu
DNA polymerase; IKBc expression was detected by immunoblot analysis using IKBa antibody Jurkat, Cos-7 and U937 cells were transfected by the DEAE -dextran method with
various plasmids - HIV- I LTR CAT HIV- I proviral DNA plasmid, plasmids encoding wild
type or mutant Tat, and wild type or mutated IKBo plasmids. HIV-I replication was analyzed
by immunoblot, mRNA analysis and p24 ELISA.
Results: TNFa and the HIV transactivator:Tat, act in synergy to transactivate the HIV LTR in
JurkatT cells. The synergistic induction of HIV LTR driven gene expression represented a
50-80 fold stirmulation and required both intact NF-KB sites and a functional Tat protein.
Co-expression of KB inhibited Tat-TNF activation of the HIV LTR in a dose dependent
mannerTransdominant negative forms of IKBat mutated in critical serine or threonine
residues required for inducer mediated (S32A,S36A) and/or constitutive phosphorylation
(S283A,T29 I A,T299A) of IKBa were tested for their capacity to block HIV LTR transactivation. An IKB(x rnolecule mutated in both N- and C-terminal sites was stable in cell lines
(TI /2>4 hours) and was able to efficiently block HIV LTR transactivation. Strikingly, transdominant nutants of IKB were at least 5 times more effective ininhibiting synergistic induction of the HIV LTR than wt IKB and also dramatically inhibited HIV- I multiplication in a single cycle infection model.
Conclusions: Tr ansdominant mutants of IKB may inhibit gene activation by two mechanisms:
I) sequestration of NF-KB proteins in the cytoplasm, and 2) direct or indirect association of
IKB with Fat, such that IKB interferes with Tat transactivation.These experiments suggest a
strategy that may contribute to inhibition of HIV- I gene expression by interfering with the
NF KB/Rel signaling pathway
Dr John H-isott, Lady Davis Institute, 3755 Cote Ste-Catherine, Montreal. Quebec, H3T
I E2 Canada.Teephone: 514-340-8260 Fax: [phone]
E-Mail: MIJHgtMUSICA.McGILL.CA
Mo.A.510
SURVEY ON HIV-I GROUP O INFECTION IN 12 DIFFERENT AFRICAN COUNTRIES.
M.Peeters, S.Mboup2, A. Gueye2, E.Liegeois, D.Patrel, M.Vanden Haesevelde3,
E.Delaportce'. Afiican Network for HIV Research. I.Laboratoire Retrovirus ORSTOM,
Montpellier; 2.Hpital Le Dantec, Daka: Senegal. 3.Innogenetics, Gent, Belgium.
Objective: To determine to what extend HIV-I group O strains are present in different
African countries

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