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

Monday, July 8, 1996

Mo.A.162 - Mo.A.270

increases from 50~-60~C over 30 min., reduced this to 4-fold. As many as 5 mutations can
be tolerated in the probe binding region with no effect on hybridization efficiency
These results indicate that subtype B isolates, which are predominant in the U.S. and
Europe, will be efficiently amplified and detected in this system given the small number of
mismatches to both SK462 and SK43 I. Similarly isolates of subtype C, D, FI, G, and H are
expected to amplify efficiently Isolates that contain 5-6 mutations (some subtypes A and E)
will be amplified less efficiently However, amplification efficiency of these isolates can be significantly improved by performing a "step up" reverse transcriptiorn, by lowering the anneal
temperature during PCR, or by incorporating unconventional bases.
C.D. Christopherson, 1145 Atlantic Ave., Alameda, CA 94501 U.S.
Telephone: [phone] FAX: [phone]
Mo.A. 162
SENSITIVE HIV ANTIGEN ASSAY DETECTING HIV- I GROUP M, HIV- I GROUP O0
AND HIV-2 CORE PROTEINS.
Louwagie, J., Garrett, PE.*, Mertens, G.~,Van Geel, A, Pollet, D., van den Abeele Anne,
Samarn E.*. INNOGENETICS NV, Zwijndrecht, B-2070, Belgium; * INNOGENETICS NV,
GENT B-9052, Belgium. ** Boston Biomedica,W Bridgewater, MA. USA. ~Belgian Red
Cross Transfusion Centre, Antwerpen, Belgium.
Objectives: To study the sensitivity and specificity of a newly developed antigen assay for
the broad spectrum detection of HIV capsid antigens in human serun and to validate the
confirmatory and immune complex dissociation reagents.
Methods: The INNOTEST HIV Antigen mAb is a sandwich ELISA. based on a human
immunoglobulin preparation as the capturing antibody (Ab) and two biotinylated monoclonals as detector antibodies.The bound monoclonals are revealed using streptavidin-peroxidase conjugate and TMB as the chromogen.The cutoff is defined as the mean of the
negative controls plus 50 mOD. Confirmation is considered positive when the signal is
decreased by at least 50% upon addition of the confirmatory antibody
Results: The specificity of the assay was estimated at 99.8% with sera from blood donors. In
supernatants from cells infected with HIV- I group M, HIV- I group O and HIV-2 viruses, the
antigen could be quantified with a detection limit for HIV- I of up to 4 pg/ml, relative to the
Pasteur p24 standard.When spiked in HIV-negative human serum, the sensitivity was I I
pg/ml. All samples could be confirmed by the confirmatory assay Each of the I 8 HIV antigen positive and both HIV antigen negative samples in the Boston Biomedica (BBI) mixed
titre panel (PRA201I) were correctly identified. Application of the immune complex dissociating reagent resulted in an important increase in signal to noise ratio for 5 samples of the
same BBI panel. In another BBI panel of 26 antigen positive seroconversions, the
INNOTEST HIV Antigen was able to detect HIV antigen earlier (2 cases) or at least equally
early (24 cases) as 4 other commercial antigen dosage assays, whereas and continuing antigenemia after seroconversion was detected later in 9 out of the 26 cases.When compared
with nucleic acid amplification methods, 23 out of 26 BBI antigen positive seroconversion
panels were found positive at the same time point and the three remairning were positive in
the subsequent sample.
Conclusion: These results indicate that the monoclonal antibody based INNOTEST HIV
Antigen detects a broad spectrum of HIV antigens and combines an excellent sensitivity
and specificity in serum samples with an efficient immune complex dissociation procedure.
Anne van den Abeele, Innogenetics, Canadastraat 2 1, B-2070 Zwijdrecht, Belgium.
Tel: [phone] Fax: [phone]
Mo.A. 163
USE OF SYNTHETIC PEPTIDES FOR DIFFERENTIATION BETWEEN HIV I GROUP M
AND GROUP O,AND FOR CLASSIFICATION OF GROUP M (A - E) AND GROUP O0
STRAINS INTO SUBTYPES
Brust Stefan*, Knapp, Stefan*, G0rtler, Lutz G**. *Behringwerke AG, Marburg, Germany;
**Max von Pettenkofer-Institut, Universitit M0nchen, Germany
Objective:To develop synthetic peptide based immuno assays to differentiate between HIV
I group M and group O infections. Furthermore to establish synthetic peptide based
immuno assays suitable to classify Anti-HIV I positive sera into subtype A - E of group M
O, and into different subtypes of group O.
Methods: Relevant epitopes from the transmembrane protein gp4 I and from the V3 loop
L region of gp I20 of different HIV isolates were identified. Synthetic peptides corresponding
> to these epitopes were synthesized and coated on micro-titration plates. Sera firom different geographic regions (Europe, East & West Africa and Asia) were analyzed by using these
u plates as solid phases for the immuno assays.
C Results:The designated combination of immuno assays based on different synthetic peptides
> was suitable to screen for HIV I group O specimen. 2 I HIV I group O infected individuals
could be identified and, in part, confirmed by PCR. Within this panel different reactivity pattern with synthetic peptides derived from different group O isolates were observed
Within the panel of HIV I group M classified sera from West Africa all relevant subtypes (A
- E) could be detected. Screening Anti-HIV I positive specimen from Europe and Asia show
C some strong reactivities with subtype B and E, additionally other subtypes.. Some of these
serologically identified samples were confirmed to be close to subtype E consensus
u sequence by PCR amplified sequences.
o:: Conclusion:The newly developed synthetic peptide based immuno assays allows the serok_ logical differentiation and subtyping of HIV I group M and group S) specimen, which is
"- most important to know doing and interpretating reliable PCR arialysis frnm newborns
O delivered from mothers infected in various geographic regions.
S. Brust, Behringwerke AG, POB 1140, D-3500 I Marburg, Germany Phone:. 49 642 I -39-2 I 64
CO Fax:. [phone] email: brust I @msmbwma.marburg.hoechst-ag.dbp.de
C
O,- Mo.A. 164
- SEQUENCING HIV ISOLATES USING THE GENECHIPTM HIV PRT ASSAY
~ Miyada, C Garrett, LiangVTran HM, Mittman M, Morris M, Kaplan P Affyrmetrix, Inc., Santa
' Clara, CA, USA
Z Objective: To develop a fast method to accurately sequence HIV isolates using a novel
X method of hybridization to high-density arrays of oligonucleotide probes.
Methods: The GeneChip HIV PRT Assay includes a high-density oligonucleotide array with
8 probes complementary to the protease gene and 242 amino terminal residues of the

reverse of the reverse transcriptase gene. A sequence of 104 I bases is determined by
hybridizing fluorescein-labeled RNA to the arrayThe array is manufactured by light-directed synthesis methods, contains over I 1,000 distinct oligonucleotide probes, and measures
1.28 x 1.28 cm.Viral samples are amplified and cloned into plasmid vectors.The recombinant HIV clones are sequenced using both the GeneChip assay and automated dideoxy
DNA sequencing.
Results: Results from the GeneChip HIV PRT Assay and automated dideoxy DNA
sequencing are compiled and compared for 12 clones.The average percentage of concordant calls per sample is 98.85% (range: 97.69-99.8 1%) and the average percentage of discordant calls per sample is I.15% (range: 0.19-2.3 1%). Reverse transcriptase drug-resistance
conferring mutations at codon 215 and neighboring codons are accurately identified in all
cases (8 samples) using either technology
Conclusion: The current configuration of the GeneChip assay sequences with an accuracy
comparable to that obtained from automated dideoxy DNA sequencing. Starting with a
single amplified sample, sequence data (104 I base pairs) is obtained within 3 hours. If steps
are batched prior to hybridization, the assay has a throughput of I 2 samples per day For
high throughput sequencing applications, the GeneChip HIV PRT Assay offers high accuracy
coupled with unprecedented speed.
C. Garrett Miyada, 3380 Central Expressway Santa Clara, CA, 9505 1, USA
Telephone: [phone] Fax: 408-48 1I-0422 Email: [email protected]
Mo.A.260
NOVEL BIOLOGICAL MEANS OF CONTROL OF HIV AND AIDS
Gallo Robert C Institute of Human Virology Medical Biotechnology Center, University of
Maryland at Baltimore, Baltimore, MD, USA
I) The C-C Chemokines (Rantes, MIP- I c, and MIP- I 3):These are a subset of chemokines
which in turn are a subset of cytokines.Their known role was in inflammation. Last year we
reported the discovery (Science, 270: 1560, 1995) that these molecules were potent
inhibitors of HIV- I and HIV-2 infection. We noted that the inhibition is virus specific (HIV- I,
HiV-2. SIV and not other tested viruses), that it worked against all tested field clinical isolates
of HIV- I but not against some CD4 T-cell line adapted strains (like Ill-B) but still effective
against other such strains like RF and MN (all in cell line cultures since 1983 or early 1984
in our laboratory).These and other results suggested that the mechanism of inhibition may
involve HIV entry a concept and new result which will be discussed in this presentation.
Our results and those of others also indicated that these molecules (Rantes, MIP-Io(, and
MIP- 113) are produced by CD8+ and CD4+ T-cells and macrophages, and that they are the
major if not only significant soluble inhibitory factors produced by CD8+ T-cells. Some have
argued against this, and repeatedly emphasized that many cytokines increase or diminish
HIV replication, and therefore, presumably there is nothing special about these chemokines.
Of course mrany cytokines affect HIV replication. For example IL-2 in promotingT-cell proliferation, will obviously promote more HIV production, whereas, other cytokines can interfere with IL-2 or with T--cell activation or other cellular events and thereby indirectly inhibit
HIV production. In this way and by numerous other possible indirect ways, it is evident that
many factors influence HIV replication by their effects on cells. These points are irrelevant to
the major issue, i.e., the C-C chemokines are the first soluble molecules - to be discovered which
directly and specifically inhibit HIV infection since the discovery of antibodies. Indeed, this
statement may be true for any virus, i.e. what other soluble specific inhibitors of any virus are
known? Moreover; in less than 6 months of our report, other groups, notably those at New
York University (Koup et. al; Nature Medicine 1996); and Zagury et. al (submitted for publication) have provided evidence that it is these chemokines which may be protective against
infection in HIV- I hyper-exposed but never infected people. I conclude that it is a moot and
irrelevant point whether the C-C chemokines are the sole or key CD8+ T-cell derived HIV
suppressive "factor" described by Levy but presumably not identified now for more than a
decade. Whether other cytokines influence virus production (long known) by effecting a
variety of cellular events is irrelevant to the discovery of novel, potent, specific naturally
occurring anti HIV molecules, which are already influencing our understanding of HIV
infection. Key colleagues in these studies are: Drs. F. Cocchi, A. DeVico, A. Garzino-Demo,
and P Lusso.
2) hCG At the last AIDS International Conference in Yokohama I reported on our model of
Kaposi Sarcoma (KS), (in vitro cultures and in vivo transplanted tumors in immune deficient
mice), leading us to conclude that KS tumors are composed of hyperplastic (non-malignant)
cells anrd at least in some cases of advanced KS, of neoplastic cells, both likely to be of
activated vascular endothelial cell origin. In the course of these studies (key colleagues in
these studies were, Drs. Y Lundari-Iskander J. Bryant, P Gill, P Hermans, O. Picard, J. Besnier,
L. Flarmmand, and D. Poretz) we found that the hormone of early pregnancy hCG, a glycoprotein heterodimer consisting of 2 chains (a and B), kills KS cells by apoptosis.This led us
to clinical collaborations and in turn to several other basic laboratory studies and will be
discussed. I will summarize the new results of both the clinical and laboratory studies in this
presentation.
RC Gallo, 725 W Lombard Street, Fifth Floor, Baltimore, MD, 2 I1201 USA Tel: [phone]
Fax: [phone] E-mail: [email protected]
Mo.A.270
PLASMA FACTORS IN HUMAN BLOOD ALTER THE ENTRY AND NEUTRALIZATION
PROPERTIES OF HIV- I: IMPLICATIONS FOR GPI20-BASED VACCINE APPROACHES
Peter L. Nara I, M. Merges I, S-C Wu2, J Spouge2. I VBS, LTCB, DBS, NCI-FCRDC, Frederick,
MD NCBI, NLM, NIH, Bethesda, MD
Conventional virology suggests one important aspect of viral tropism/pathogenicity is mediated by specific virus ligands interacting with specific cellular receptors. Factors, however,
which govern and/or influence the specificity of these interactions within the host may
involve colloidal or soluble molecules capable of additional enzymatic or cooperative interactions.\We have reanalyzed the immunobiology of field isolates and laboratory strains
under more relevant physiologic and/or cellular conditions in an effort to determine if other
factors exist which could provide new insights into HIV- I entry than might be currently
appreciated. Primary cultures of either peripheral blood mononuclear cells (PBMC's) or
blood-derived macrophages (BDM) in the presence of physiologic concentrations of normal
human blood plasma were found to enhance the infectivity of primary isolates by recruiting
otherwise non-infectious particles in a first order and time dependent wayThis enhance