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

Track A: Basic Science

Tu.A.2101 - Tu.A.2106

Conclusions: We demonstrated that TCA3 and IL- I 2 expression plasmids enhanced the
HIV I specific CMI induced by DNA vaccine. Direct inoculation of these expression plasmids in muscles would be one of the ideal adjuvant strategy in HIV vaccine development.
FTsuji, 3-9 Fukuura, Kanazawa ku, Yokohama 236, Japan Telephone: [phone] Fax: 81 --15 /87 2509
Tu.A.2101
ANTIBODIES IN HUMAN SERATO HIGHLY CONSERVED EPITOPES INTHEVl/V2
DOMAIN OF gpl20 THAT MEDIATE POTENT NEUTRALIZATION OF
MACROPHAGETROPIC HIV- I ISOLATES.
Pin ter Abraham, WJ. Honnen, Z. Wu, O.Troshev, S.C. K ayman. Laboratory of Retroviral
Biology: Public Health Research Institute, New York, NY 10016.
Objective: To identify epitopes in the HIV-I envelope proteins that mediate potent neutralizationr of primary rn acriophage-tropic (M-tropic) viruses by antibodies present in some
human sera.
Methods: We have identified human sera with potent neutralizing activities for a number of
M-tropic isolates.These sera contain crossreactive antibodies that recognizeVI/VN2 domains
friom a broad range of isolates, including foreign Glade B and Thai lade E isolates.These
antib odies were isolated by affinity chromatography using recombinant fusion proteins that
exp ressed native V I/V2 domains, their epitope specificities were characterized, and their
neutralizing actities for a number of M-tropic primary viruses was tested.
Results: Depletion of anti-V I/V2 antibodies fiom several sera resulted in a significant
decrease in neutralizing titers fior M-tropic viruses.VI/V2-specific antibodies eluted from the
columns hrd potent neutralizing activities for several M-tropic viruses. Antibodies with distinct epitope specificities were fractionated from one serum by sequential elution of the
column with low pH buffers followed by 5M Guanidine-HCI.Type-specific antibodies eluted
in the ear liest fra cteions while ncrossreactive anti-V I/V2 antibodies required more stringent
elution conditions. Potent neutralizing activity for heterologous primary viruses was present
moainly in the GuHCI fiacti on.The activity of these antibodies for some primary viruses was
remarkable, and higher than that obtained with the most potent monoclonal antibodies;
e.g., the ADA strain was neutralized with an ND50 of -3 ng/ml and an ND95 of-50 ng/ml.
Conclusions: The potent neutralizing activity of anti-VI/V2 antibodies in human sera for
heterologous primary viruses indicates that the V I/V2 domain contains highly conserved
epitopes that are sensitive neutralization targets for HIV-I in vivo.The effective depletion of
neutralizing activity for M-tropic viruses from human sera by passage over a V I/V2 affinity
column further indicates that the V I/V2 region is a major neutralization domain in primary
viruses, and suggests that vaccines that induce antibodies against such V I/V2 epitopes may
provide effective protection against HIV-1I.
A. Pinter, Public Health Research Institute, 455 First Avenue, NewYork NY 10016.Tel: (212)
578 0879; Fax: [phone] e-mail: pinterphri.nyu.edu
Tu.A.2102
DEVELOPMENT, PRECLINICAL,AND CLINICAL TESTING OF CANDIDATE AIDS
VACCINES
Walker, Mary Clare, McNamara J, Schultz AVogel F, Bradac J, Sawyer L, Miller N, Glass M,
Savarrese B, Wescott S, Flores J, Lawrence D, Grabowsky M, Fast P VPRP DAIDS, NIAID,
NIH, Bethesda, MD, USA
Objective: To encourage and support the development of a safe and effective AIDS vaccine(s) through basic and applied research, including testing in animals and humans, is a
major-goal of the NIAID Vaccine and Prevention Research Program (VPRP).
Methods: The correlate(s) of immune protection against HIV-I are unknown. It is anticipated that both vaccine-induced humoral and cellular immune responses may be required for
protection. As HIV is transmitted sexually across mucous membranes, a vaccine may also
need to induce protective responses at mucosal sites where transmission occurs.
Additionally vaccine-induced immunity should be equally effective against all genetic subtypes of HIV.Therefore, the NIAID VPRP supports the evaluation, through integrated fundamental research and empiric development, of a variety of vaccine concepts in preclinical
and clinical trials. It also supports studies of the genetic and antigenic variation of HIV to
facilitate vaccine design. A network of domestic and international clinical sites to conduct
trials of candidate AIDS vaccines and other prevention interventions and provide education
about AIDS and its prevention are in place.
Results: About 20 candidate AIDS vaccines of the more than 50 in development, are
already in Phase I trials worldwide. Most are subunit HIV env gene products designed to
induce anti-HIV antibodies. Other products are engineered to induce cytotoxicT cell (CTL)
activity or mucosal imnmunity; these are based on gag as well as env genes and are delivered
as live recombinant constructs, lipopeptides, or in encapsulated or particulate form. Newer
candidates are based on mrore than one HIV gene or on multiple isolates of HIV.
Combinations of different types of products: e.g., live recombinant plus subunit, are being
tested in animal models and in humans to determine if responses may be augmented or
broadened. Close to Phase I trials, are such immunogens as virus-like particles (pseudovirions) and nucleic acid vaccines. Preclinical studies of whole inactivated HIV and of attenuated
live SIV and HIV prototype vaccines are underway.
Conclusions:The development of an AIDS vaccine(s) requires basic and empiric laboratory,
animal and human research in partnership with industry and the communities where a candidate vaccine will be tested and if efficacious, used.
Mary Clare Walke, CDB/DAIDS/NIAID/NIH, Solar Bldg., Room 2A29, 6003 Executive Blvd.,
Bethesda, MD 20892-7620, USA [phone]; [phone]
Tu.A.2104
V3 MULTI-EPITOPE POLYPEPTIDES.THE NUMBER OF V3 REGIONS AFFECTS THE
EXPOSITION OF EPITOPES AND THE IMMUNOGENICITY
Menndez A, Montero M, Quintins D, Gomez C.* Naver L. Vlarubia D. Carpio F, Hayes,
C) and Duarte C. Center for Genetic Enginuerirug and Biotechnology and *National
Laboratory for AIDS, Havana, Cuba
Objective: To generate Multi Epitope Polypeptides (MEPs) carrying different number ofV3
regions and to compare the antibody response induced by these proteins in rabbits and
mice.

Methods: The MEPsTAB9 and TAB 3, expressing 15 amino acids of the V3 loop from different HIV-I isolates were constructed.TAB9 included strains LRI50 (sequence numbered 150
in La Rosa et al, Science 234, 1990), JY I, MN, PF, BRVA, and LAI, while in TAB I 3 two additionalV3 regions with the central tetrapeptides GPGQ and GQGQ were added. Both proteins were expressed as inclusion bodies in E.olr fused to a fiagment of a N.rreningitrds
outer membrane protein (OMP).They were purified and the surface exposition ofV3 epitopes from strains MN, LAI and JY I was evaluated using specific monoclonal antibod ies
(Mabs). Four New Zealand rabbits and ten Balb/c mice were immunrsed with the MEPs
emulsified in Freund's Complete Adjuvant. After four doses the sera were evaluated in
ELISA using MEPs and conjugated V3 peptides.The " nvtro" neutralizing capacity against
four HIV- I isolates was also measured.
Results: Both proteins were expressed at high levels in E.coli and readily purified. When the
reactivity of the MEPs with Mabs specific for common V3 epitopes was compared in ELISA
we observed significantly higher values forTAB9 in every case. Similar results were achieved
in a sandwich assay with a combination of two Mabs. Both proteins were immunogenic in
mice and rabbits, but antibody titers against MEPs and synthetic peptides were alway higher forTAB9, as well as the frequency of response against peptides. Strong neutralizing activity against isolates MN, LAI, JY I and RF was detected in one out of fourTAB9 rabbit sera,
and against isolates MN and LAI in one out of fourTAB 3 sera.
Conclusions: The MEPs evaluated in the present study, fused to a fiagment of a N.nrenig,tidis antigenic OMP were immunogenic in rabbits and mice.The addition oftwoV3 regions
to the MEPTAB9 resulted in a lower surface exposition of V3 epitopes and in a lower antibody response to the protein.
A. Menendez,Vaccine Division, CIGB, PO.Box 6162, Cubanacan, Havana 10600, Cuba Phone
53-7-218070 Fax [phone] email: guillen @ingen.Cigb.edu.cu
Tu.A.2105
ASSEMBLY AND IMMUNOGENICITY OF HIV-I CORE/ENVELOPE CHIMERIC
PROTEINS EXPRESSED IN BACULOVIRUS- INFECTED CELLS.
Truong Catherine*, Brand D*, Roingeard P*, Mallet F**, Brunet S*, Barin F5. *Universite F
Rabelais,Tours, France; **Ecole Normale Superieure, Lyon, France.
Objective:To study assembly and immunogenicity of recombinant HIVI gag polyproteins
expressing neutralizing epitopes of HIVI gpSU.
Methods: Several chimeric genes have been constructed by an original procedure using the
polymerase chain reaction procedure, and then cloned in a baculovirus transfer vectorThe
V3 consensus sequence (subtype B) or the conserved C4 region of the CD4 binding site
(CD4bs) have been inserted in place of two exposed epitopes of the p24 region (aa 196 -226 and 303-317). An unmodified gag gene (wild type) was also cloned following the same
procedure. After co-transfection of the recombinant vectors with wild type baculoviral
DNA, recombinant viruses have been selected and purified. Auto-assembling into virus-like
particles has been studied by electron microscopy examination. Immunogenicity of the
chimeric proteins was evaluated in mice. Mapping of antigenic sites was done with sera
from immunized mice and peptides overlapping the entire Gag precursor.
Results and Conclusion: Wild-type p55 and chimeric core/envelope proteins (p55-V3, p55 -CD4bs) derived from insertion at positions 196-226 were capable of auto-assembling.
Chimeric proteins derived f-om insertions at positions 303-317 did not assemble into
immature particles, but were erasbedded into spherical structures that derived probably
from the plasma membrane of infected insect cells.The response to env epitopes was weak
and no neutralizing activity was detected in mice sera. Mapping of the gag epitopes showed
that p24 and p I7 epitopes presented to the immune system differed from the mature form
(p24 or p 17) and the immature form (Pr55), but also were dependent of both the nature
and the site of inserted sequences
CTruong, laboratoire de Virologie, CHU Bretonneau, 37044 Tours Cedex, France.Tel. (33)
47474747 ext. 34086 - Fax. [phone]
Tu.A.2106
IN VIVO ANALYSIS OF INOCULATED rBCG-HIV CANDITATE VACCINE:
LOCALIZATION AND IMMUNE RESPONSES AGAINST HIV-PND ANTIGEN
Nakasatomi T-"2, Haga S3), Shibata S4), Naganawa S2), Someya K2), Ando 52),Yoshizaki
H2), OkamotoY2),Taki M1), Kitamura K2), Miyamoto K2),Watanabe K2),Yamada K ),
Yamazaki S2), Honda M2). I) St. Marianna Univ. School of Medcine, 2) AIDS Research
Center, NIH, 3) Department of Bacteriology, NIH and 4) Department of Veterinary
Medicine, NIH
Objective: To elucidate the in vivo expressions and dynamics of the HIV PND antigen for
the understanding of the mechanisms of immune induction to the viral antigen by recombinant BCG-based vaccine for HIV I.
Methods: We constructed the recombinant Mycobacterium bovis BCG (rBCG)-based vaccine which inserted HIV principal neutralizing determinant (PND) genes among the (a-antigen of Mycobacterium. Guinea pig were inoculated the rBCG intradermally or olally Eighth
weeks latenr, these animals were skin tested by intradermal injection of HIV PND antigen
with KLH. Local skin reactions were measured 24h after challenge. Serum antibody titers for
the HIV-PND antigen was measured by peptide based ELISA.The tissue, such as the local
skin sites, inguinal regional lymphnodes, spleen and peyers patch, were removed adequately
and were studied by the PCR for a chirneric protein of auantigen and PND-peptide, the
histological staining of rBCG, the colony count of live bacteria and the pathological examination.
Results: The binding activity titers to PND peptide were elevated after inoculation.
Induction of HIV specific delayed type hypersensitivity reaction were observed in guinea pig
with the rBCG. By intradermal inoculation of rBCG to guinea pigs many lump of the rBCG
wan detected in the local skis sites week after the noculatie. Fur ther the numder of the
barcili was decreased te few in the local ski eiaes hy histelugical saising. Is she inguiI
regional lysphnode, it wan detected until 6weeks after inoculatrer but thereafter it was
set detected. All the ether tinssuin tested was rnegatiye fur the hacilli, such as lung spleen
and peripheral blood.The rBCG was detected mainly local skin sites and small numbe of
bacilli were also found in inguinal lymphonodes.The inoculated rBCG by orally induced
humoral immune response and observed in the peyers patch.,,D
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