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

TRACK A: BASIC SCIENCE

TuA 0500-TuA 0505

TuA 0500 MACACA NEMESTRINA: A NEW PRIMATE MODEL FOR HIV-1
INFECTION. Agy. Michael B; Frumkin L**; Corey L**; Coombs R**; Wolinsky S***;
Morton W*; Koehler J**; Katze MG.*,**. *Regional Primate Research Center,
**School of Medicine,.University of Washington, Seattle,WA; ***School of
Medicine, Northwestern University, Chicago, IL.
Obiective: To develop a useful animal model for HIV- I infection in a macaque species.
Methods: Macaque PBMC from 3 species were purified from ficoll, PHA stimulated for 72 hours, and
infected by HIV-1L AI or SIVmne. Further, M. nemestrina PBMC from several animals were similarly
prepared and infected by 2 additional HIV-1 strains. Viral replication was monitored by antigen capture
ELISA or radioimmunoprecipitation (RIP) analysis. Thin sections of infected M. nemestrina lymphocytes
were examined by electron microscopy (em). Two animals were subsequently inoculated with autologous
lymphocytes infected by HIV-1LAI. Blood was collected at regular intervals for PBMC coculture, and for
serological, and PCR analyses.
Results: HIV-ILAI replicated in M. nemestrina CD4 positive PBMC, but not in PBMC from M.
fascicularis or M. mulatta. SIVmne replicated equally well in all 3 species. HIV- ILAI, HIV-INIA-3, and
HIV-IJR-CSF replicated in cultured M. nemestrina PBMC. RIP analysis revealed denovo HIV-1 protein
synthesis in infected M. nemestrina PBMC. Cell associated and budding virions were seen by em. HIVI was recovered intermittently from both infected animals between 2 and 24 week post-inoculation (p.i.).
Both animals produced a sustained antibody response to a full range of env and gag gene products
through 72 weeks p.i. HIV- I DNA was demonstrated in PBMC from both animals by PCR through 52
weeks p.i. Subsequent in vivo studies have confirmed these observations.
Conclusions: This model provides a novel means for studying pathogenesis of acute HIV-1 infection
and a means of evaluating comparative effectiveness of various candidate HIV-1 vaccines and anti-viral
drugs.
Michael B. Agy, Ph. D., Regional Primate Research Center, SJ-50,
University of Washington, Seattle, WA, 98195, USA. Telephone:
1- [phone], FAX: 1- [phone].

TuA 0501 INFECTION OF SIVMc-CHIMERIC VIRUSES HAVING HIV-1 ENV GENE TO MACAQUE
MONKEYS. Hayami, Masanori; Shibata, R.; Sakuragi, S.; Igarashi, T.:
Adachi, A. Institute for Virus Research. Kyoto University, Kyoto, Japan
ObJective: As HIV-1 is able to Infect only higher primates, construction of chimeric
viruses between HIV-1 and SIVkeo infectable to macaque monkey was attempted to
establish HIV-l-macaque monkey Infection system.
Methods: A series of chimeric viruses were constructed in vitro between HIV-1 (NL 432)
and SIVlo (239) infectious DNA clones. Some of the chimeric clones which showed
growth capability in monkey PBL were Inoculated to cynomolgus (macaque) monkeys and
their infectability was assessed by antibody response, detection of virus genome and
virus isolation.
Results: A chimera (NM 3) carrying the LTR, gag, pol, vif and vpx of SIVMo and tat,
rev, vpu and env of HIV-1 was replication competent In monkey PBL. In contrast, a
chimera (Nh-1) carrying vpx, vpr, tat, rev and env of SIVm.,o and LTR, gag, po, vi, f of
HIV-1, and a chimera (NM-2) carrying rev and env of SlVMAo and gag, poi, vif, vpr, tat
of HIV-i could not grow in monkey PBL. NM-3 was Inoculated to 2 macaque monkeys and
proved to be infectious. The other chimera NN-3n, a modified form of NM-3 which has
Intact nef of HIV-1 was also infectable to macaqne monkey. The construct of reisolated
viruses was the a some as that of the original construct. Sofar, no sign of disease
onset was seen in these monkeys.
Conclusion: These data indicated that the sequences Important for macaque cell
tropism lie within the LTR, gag, pol and vif sequences of the SIVMAo genome. The
successful infection of macaque monkeys with chimeric viruses containing HIV-I env
gene may facilitate development of vaccine based on HIV-1 env, because they can be
used as chllenge virus to macaque monkeys, instead of chimpanzee.
HAYAMI, Masanori. Institute for Virus Research, Kyoto University,
53 Shogoin-Kawaharamachi, Sakyou-ku, Kyoto 606, Japan Telephone;81--7-751--3982
FAX; 81-75-761-9335

TuA 0502 SERIAL IN VIVO PASSAGE OF SIV FROM INFECTED MACAQUES TuA 0503 SIV-related malignant lymphomas express
WITH END STAGE VIREMIA RESULTS IN DRAMATIC INCREASE IN transforming EBNA-like proteins and B-cell activation antigens. H.Feichtinaqer'*;
STRAIN VIRULENCE Holterman. Lennart; Niphuis, Henk; Dubbes, Rob; S.LU", I.Embrg"', E.E.Kaaya"**, P.Putkonene", G.aberfeldh"", P.Bibrfeld"; *Dep. aof
Koorstra, Wim; MurphyCorb, Mickey; Heeney, Jonathan; Dept. of Chronic Pathology, Univ. of Innsbruck, Austria; "lmmunopathology Lab., Karolinska HospitalAnstitute,
Koomstra, Wim; Murphy-Corb, Mickey*; Heeney, Jonathan; Dept. of Chronic **'Dep. of VK-ology and..Imrmunology, National acteriological Laboratory, Stockholm, Sweden;
and Infectious Diseases, ITRI-TNO, Rijswijk, The Netherlands; *Dept. of
Virology, Delta Regional Primate Center, Covington, Lo., U.S.A.

Obiective: To determine if repeated passage of SIV in vivo results in increased virulence as
measured by the progression time to AIDS.
Methods: Ten age matched juvenile Macaca mulatta were infected with an equivalent dose of
SIVB670. The first animal to develop AIDS (7 months) was the donor of 2 x 106 PBMC or lymph node
filtrate as a source of virus. Subsequent passages were performed by passing uncultured PBMCs
(2 x 106) from the first individual to develop advanced disease and viremia. Complete hematological
and pathological analysis was performed on each animal with specific emphasis on helper/memory
(CD4/CD29) cell loss and persistent plasma viremia as measured by specific SIV antigen capture.
Results: Each consecutive passage resulted in a dramatic reduction in time to development of
terminal disease characterized by rapid helper memory cell loss and high, persistent levels of plasma
antigen.
Conclusion: Based on the hypothesis that the most virulent virus types evolve and are predominant in
the final stages of disease development, we have passaged PBMCs from macaques which develop
terminal viremia and disease most rapidly. Consecutive in vivo passage of virus and selection in this
manner has resulted in a virus strain capable of eliciting a rapid disease course to AIDS in one to two
months. Characterization of this strain will assist us in understanding the pathogenic viral determinants
of disease.
Holterman, Lennart, ITRI-TNO, Rijswijk, Holland;
Telephone [phone]; FAX [phone]
TuA 0504 NEUTRALIZATION OF PRIMARY HIV-1 ISOLATES BY SERA FROM PRIMATES
IMMUNIZED WITH RECOMBINANT HIV-SF2 GP120. Haiawood. Nancy*; Yoshiyama,
H."; McClure, J.*"'; Ho, D.D."**; Steimer, K.S'. *Chiron Corporation, Emeryville, CA,
"**Aaron Diamond AIDS Research Center and New York University School of Medicine,
New York, NY, "'***Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA
Obiectives: Effective immunization of experimental animals, and ultimately humans, with subunit vaccines against
HIV-1 will require the induction of broad-spectrum immune responses. Neutralizing antibodies induced by subunit
immunization were examined in an effort (1) to determine the capacity of these sera to neutralize laboratory and
primary HIV-1 isolates and (2) to understand the mechanism for induction of these antibodies.
Methods: Baboons were immunized with recombinant gpl20 produced in CHO cells as a native, glycosylated
antigen (rgpl20) or in yeast as denatured, nonglycosylated protein (env 2-3) as described (Haigwood et al, 1992. J
Virol 66: 172). Sera from all 51 animals were analyzed for neutralization of laboratory isolates and characterized for
V3 region specificity and CD4-blockidng activity; the 15 highest titer neutralizing sera were analyzed for
neutralization of primary isolates of HIV-1. Primary HIV-1 isolates were either (1) obtained directly from infected
human peripheral blood mononuclear cells (PBMC) by stimulation with CD3 antibody or (2) grown for a single
passage in human PBMC. Neutralization was assessed in human PBMC in short term culture.
Results: Baboons immunized with rgpl20 had neutralizing antibodies effective against 10 laboratory isolates,
including two African isolates, while those immunized with env 2-3 neutralized only HIV-SF2 and rarely HIV-MN
(Haigwood et al, ibid.). Blocking studies with V3 peptides indicated that neutralization of non-African isolates was
due, in part, to better presentation of the principal neutralizing determinant (PND) by rgp 20 than by env 2-3. As
seen with the analogous fraction from HIV antibody-positive human serum (Steimor et al., 1991. Science 254:
105), the conformation-dependent antibody fraction from rgpl20-immunized baboons has the broad neutralizing
capacity and blocks the binding of HIV-SF2 gp120 to CD4. Unfractionated sera from these same animals also have
titers against several primary field isolates. Analysis of V3 region sequences from these viruses shows that the
primary V3 sequence is not necessarily predictive of ability to neutralize. These results suggest that neutralization
via binding to V3 is unlikely to be the sole mechanism involved. Expanded studies are underway to determine the
neutralizing capacity of these sera against a panel of approximately 20 primary isolates from geographically distinct
North American sites.
Conclusions: Native rgpl20 subunit immunization can induce cross-neutralizing antibodies effective against both
laboratory and field isolates of HIV-1 that are directed both to the PND and to conformational determinants. A
vaccine based upon rgpl20 from the SF2 isolate may thus be effective against a broad range of HIV-1 isolates.
Nancy L. HAIGWOOD, Chiron Corporation, 4560 Horton STreet
Emeryville, CA 94608 USA; tel: [phone]; FAX: [phone]

Objectives: Characterization of the lymphoma subtype and demonstration of the pathogenic importance of a
cynomolgus E-ymphotropic herpes virus (CBLV) in SIV-related lymphomas of cynomodgus monkeys in analogy
to corresponding EBV-assodated AIDS lymphomas
Methods: 33 cynomolgus monkeys were infected with simian immunodeficency virus (SIVsmm3). Presence
of CBLV in lymphomas was assayed by Southern blotting with EBV-specific probes. Typing of EBNA-like
proteins was done by Western blotting (WB) of protein lysates from lymphomas (13) and cultured lymphoma
cells (3) using human reference and monkey sera as well as poly- and monovalent antibodies against different
human EBV-associated proteins. Expression of B-cell activation antigens was studied by immunohistochemistry
with rossreaive monodonal antibodies (mabs) against dustered human leucocyte antigens.
Results: 13/33 SIVinfected monkeys developed malignant lymphomas. Most of these lymphomas showed
DNA-sequences crosshybridizing with probes specific for human EBV. Western blotting revealed the presence
of an EBNA-2 like molecule in protein lysates from all lymphoma biopsies and cultured lymphoma cells. This
finding was confirmed by immunohistochemical demonstration of this nuclear protein in biopsies and cultured
cells. Furthermore, a protein similar to EBNA-1 could be demonstrated in 7 tumor biopsies and 3 cell lines. Other
potentially transforming proteins associated with human EBV indluding latent membrane protein (LMP) were not
unequivocally identifiable. The immunephenotype of the lymphomas showed a pattem in accordance with
adivated B-cels with expression in lymphoma cels of CD23 and occasionally CD30.
Conclusons: The association and expression of EBNA-like molecules and B-cell activation antigens of
malignant lymphomas in SIVsrmm3-infected cynomolgrs macaques suggests their sirr'larity to a major subtype
("LCL-lke" type) of EBV-related AIDS lymphomas
Dr.Hans Feichtinger, Dep.of Pathology, Univ. of Innsbruck
Mellerstr.44, A-6020 Innsbruck, Austria
Tel.: [phone] Fax: [phone]
TuA 0505 VERY BROADLY NEUTRALIZING HUMAN MONOCLONAL ANTIBODY
(HuMAb) AGAINST THE CD4-BINDING SITE OF HIV-I gpl20
Tilley, Shermaine A.*, Honnen, W.J.*, Racho, M.E.*, Hilgartner, M.**, & Pinter, A.*
*Public Health Res. Inst. & **NY Hospital-Comell Med. Center, NY, NY
Objective: To develop HuMAbs against HIV with potent, broadly neutralizing activity.
Methods: HuMAb 5145A was derived by EBV-transformation of PBMC from an asymptomatic, seropositive
hemophiliac and characterized essentially as described for HuMAb 1125H [Tilley et al. (1991) Res. Virol.
142:247). Neutralization and synergistic neutralization analyses were performed using - 5 x 104 infectious
units of virus as detailed [Tilley et al. (1992) AIDS Res. Human Retroviruses, in press].
Results: A new neutralizing HuMAb, 5145A (yl, ic), is against a conformational epitope overlapping the
CD4-binding site of HIV-1 gpl20; it possesses broader HIV-1 strain reactivity than any mAb yet described
against this epitope cluster. Immunofluorescence analysis shows that 5145A strongly recognizes all HIV-1
strains tested thus far. This includes 4 North American (NA) strains (MN, SF-2, IIIB, RF) and 10 Central
African strains. The V3 loop of each of the African strains has been sequenced, and these data show that each
of the strains is unique and that their V3 sequences are more highly related to the African V3 consensus
sequences than to the NA V3 consensus sequence, as expected. 5145A does not recognize LAV-2, an HIV-2
strain. The breadth and potency of 5145A in neutralization assays against the 4 NA strains listed above is
greater than or equal to that of our previously described potent, neutralizing HuMAb against CD4-binding site,
i.e., 1125H. These assays show that 50% of each of the NA strains is neutralized by -1 tg/ml of 5145A. and
that over 95% of each virus is neutralized by 10 lg/ml of 5145A. 5145A's potency against IIIB is similar to
that of the type-specific anti-V3 loop mouse mAb, 0.50. When combined with 4117C (anti-V3 loop HuMAb)
at a 1:1 ratio, 5145A exhibits synergistic neutralization of the MN and SF-2 strains; the level of synergism
observed is comparable to that previously observed with 4117C paired with 1125H [ Tilley et al., AIDS Res.
Human Retroviruses, in press]. Epitope mapping of 5145A is in progress.
Conclusions: A very broadly neutralizing HuMAb against the CD4-binding site of HIV-1 has been developed.'
This HuMAb exhibits potent synergistic neutralization of HIV-1 when paired with a neutralizing anti-V3
HuMAb, 4117C. HuMAb 5145A has excellent potential as a passive immunotherapeutic against HIV-1.
Dr. 5ertaine_ A. T 7le
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