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

Tuesday, July 9, 1996

Tu.A.163 - Tu.A.261

Results: Among three HT LV ELISA positive samples, one was WB pos
remainders were indeterminate but closely related.Viruses were isolat
chimpanzee. Results of PCR using primers for pol, tax regions were su
netis correlation with HTLV II.
Conclusions: HTI V-infections appear to be endemic among African
Amnerinrdiains according to a number of recent studies. Here we report
5 LV II, the counterpart of HTLV II, from an Old World non-human pr
ies of the proviral DNA should give new insight toward understanding
demiology and evolution of PTLVs and human migration.
[).A. McLeod, A/Chief, Nat.Lab. for Viral Oncology, Bur. of Microbiology,
Disease Control,Virus Bdg. 100 1A I, Tunney's Pasture, Ottawa, Ont. KI
8178 ax: [phone] email:[email protected]
Tu.A. 163
SPONTANEOUS LYMPHOCYTE PROLIFERATION AND IL-2 PRO
HTLV-II INFECTED VENEZUELAN AMERINDIANS.
Baroja, M.l., Leon Ponte M,Naya O*,Bianco NE, Echeverria de Perez,
Immunology and *Tropical Medicine, Faculty of Medicine, Central Univ
fircas, Venezuela.
Objective: To analyze Peripheral Blood Mononuclear Cell (PBMC) res
HT I V II infected Venezuelan Armerindians.
Methods: Ten (10) HTLV-II infected and 12 uninfected Guahibo Amer
were studied. Fresh PBMC were isolated and cultured in either mediu
Lymrphocyte proliferation was measured by 3H thymidine incorporati
2 production were supplemented with the anti Tac mAb to block IL-2
vent II 2 consumrption. IL 2 activity was measured using a bioassay on
Results:

itive for HTLV II, the
ed from the positive
ggestive of phylogenatives as well as
t the first isolate of
rimate. Detailed studof molecular epiLab. Centre for
A OL2 Tel: (61 3) 957 -
analysis of 720 bp of the LTR performed in 1I0 Spanish samples showed that all these samples belonged to lb subtype with a divergence of 7.5% and I1.66% compared to MoT (lla)
and NRA/G I 2 (lib) isolates, respectively RFLP analysis demonstrated the presence of the
IIb4 subtype restriction pattern in 26 samples, a lib5 subtype pattern in one Italian injecting
drug user, and a Ila0 subtype pattern in 2 Italian samples (blood donors), according to
Switzer's nomenclature (SwitzerWM, et al. J Virol 1995;69:621 -632).This is the first report
of the presence of IIb5 in Southern Europe and IIa0 among Italian blood donors. RFLP correlated with nucleotide sequence and phylogenetic data obtained in this study demonstrating the ability of the RFLP method to predict the phylogroup of HTLV II-infected samples.
Dr Alejandro Vallejo. FDA/CBER. HFM-315. 140 I Rockville Pike. Rockville, MD 20852, U.S.A.
[phone]

Tu.A.260
IDENTIFICATION OF PROTEASE INHIBITORS CONTAINING ALLOPHENYLNORSTADUCTION IN TINE ACTIVE AGAINST BOTH WILD-TYPE AND KNI-272-RESISTANT HIV-I VARIANTS
Tanaka M*, Mimoto T, Anderson B*, Gulnik S#, Bhat,TN#,Yusa K*, Hayashi H, KisoY~,
Gloria. Institutes of Erickson JW#, Mitsuya H*. *National Cancer Institute, Bethesda, Maryland, #SAICversity of Venezuela, Frederick, NCI-FCRDC, Frederick, Maryland, USA; Japan Energy Co.,Tokyo, Japan; ~Kyoto
Pharmaceutical University Kyoto, Japan.
ponses in a group of Objective: HIV I develops in vitro a high degree of resistance to KNI-272, a substrate based,
peptide-mimetic HIV protease inhibitor containing allophenylnorstatine (Apns), by acquiring
indians (controls) mutations in the protease-encoding gene.We synthesized various Apns-containing protease
m, PHA or IL 2. inhibitors and identified several compounds showing activity against both wild type and
on. Cell cultures for IL- KNI-272-resistant HIV I.
2 receptor and to pre- Methods: Forty-four Apns- containing inhibitors were tested against HIV- I LAI' KNI 272-resisCTLL. tant HIV-I (HIV 1272R, and various infectious HIV I clones containing subsets of mutations
conferring KNI-272-resistance.The IC50 values of KNI 272 against HIV I LAI and HIV I 272R
were 0.04 pM and 2 pM, respectively
U CTISN mU/mi2 Results: Of 44 Apns-containing inhibitors, KNI-24 I and its related compounds showed
edium PtHA activity against both HIV- I LAI and HIV-1272R in vitro. Among the inhibitors which proved to
(0.5 ug/ml) be active against HIV- I HXB2D and KNI-272-resistant infectious HIV I clones, KNI-24 I was
48+77* 5883 4 358 most potent (see Table). Inhibition constants (Ki) determined using recombinant mutant
30+ I 6681 +961 proteases corroborated the differential activity of these inhibitors against HIV- I HXB2D and
KNI-272-resistant clones. X-ray crystallographic analysis and modeling studies of enzymeinhibitor complexes were also performed.

SYI t Y IT PROIf hRAl ION
ipot Cr cursi s,2 i'') I 2 2 P
1 / (10 U/1711) 0.5
ttl I+l 1833t 8 I* I ) _~ ', 38* 4058~1 10 2298
(c irfh. i L 30 103 999-253 3/36~4 5 5 215
IRstlts ire epressed in cpm (media+S) *p<0.05. *p<0.025
211 ) activity was measured on supernatants collected after 48h:

IL 2 PRODL
HA M
3+5703 1

Conclusions: Our results showed significant differences in HTLVII infected individuals when
compared to non ca-rriers regarding: I. Increased Spontaneous Lymphocyte Proliferation
(SLP) at daiys 5 and 7.2.- Increased spontaneous IL-2 production.The proliferative response
of PBMC from the HTLV II infected group to either PHA or IL 2 (day 3) was similar to conrols. HIowever, in two HTLV-II carriers, a correlation was observed between the highest SLP
iat dys anid 7 and the response to exogenous IL 2.These results suggest a pre-activated Tcell status probably related to the HTLV-II infection.
C. FCheverrfa de 'Perez, Aerocav 1216, PO. Box 02-5304, Miami, FI 33102-5304.
Iel: 58-2 605.3429 Fax: [phone] I.
Tu.A. 164
MOLECULAR CHARACTERIZATION OF HUMAN LINPHOTROPIC T II TYPE(HTLV-I1I)
SUBTYPE A VIRUS IN A PATIENT INTRAVENOUS ADDICT WITH AIDS.
'Bili rne1, Biglionre.) "Weisburd, G. "Avila, M, 'Libonatti, O, "Arbulu,M, "'Gessain, A. 'National
Center of Reference for AIDS, Microbiology Dep, Fac.Medicine,UBA-"Infectious Diseases
hervice,Hosp. ( Car rasco, Rosario, Argentina-" U.D 'Epidem. Onc.Virus, I. Pasteur, Paris
Introduction Very few isolation of H lTLV -I I virus were reported in the world that same as
the I It LV I in drugs users and aborigines. Moleclar studies allow to classify 2 types: the
A(T ILV-I I II MO) and thIe B(HTLV II-NRA and I2). Previus studies demonstrated the
presence of the T LV-I IB in tobas and rnatacos of the Argenitnian north. In this work is
described the molecular characterization of the HTLV- I I subtype A, in a injection drug user,
with AIDS, inpatient in our Service Infectius Deseases, Hosp. Carrasco, Rosario.
Material and Methods: In 1993,1I0 patient were studied young adults, I female and 9 masiculine it radom,having all conduct of risk of intravenous drug userThey were carried out
serologicral detection, confirmation, in vitro amplification, clonation and study of sequence
with moe requrent mrethods.
Discussion and result: Of the 10 studied patient I was positive for the HTLV-I I (N~ sample
8) confirming the presence of virus by means of PCR.The amplified product was a region
of 590 pb of the gene of the gp2 I transmembranaria of the cover using WH I WH3 like
external primers and WH I WH-2 like internal primers.The cloned in vectorial Bluscript and
the posterior secuenciation of this region confirmed that the studied virus belong to the A
sultysi' of the HTLV-I I.
Conclusions: By the used methods remain demonstrated the presence of the HTLV -I I,A
s rtype, in injection drugi user with AIDS, being this letter the first description in our country in,patient wil these characteristic.

Cloned HIV-l
HIV I xur>
HIV- I v3
HIV 71/ 4,,
I- IV........

ICst (pM)
KNI-272 KNI-208 KNI-223
0.01 0.04 0.09

KNI-241
0.02

KNI-303

0
0:>
o
u)
5:
n
ia
0
C
is
Qs
C
cO
U
0
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a
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220

Conclusions: We identified several Apns-containing protease inhibitors which are active
against both wild-type HIV I and variants resistant to KNI-272. Using these inhibitors as
lead compounds and X ray crystallography data of enzyme-inhibitor complexes, the design
of new types of protease inhibitors, which tightly bind with the wild-type and mutant
enzymes and delay or block the development of certain protease inhibitor resistant HIV-I
variants, may become possible.
Masatoshi Tanaka, M.D., Ph.D.The Experimental Retrovirology Section, Medicine Branch,
National Cancer Institute, Bldg 10, Rm 5All, 9000 Rockville Pike, Bethesda, MD, USA.Tel:
30 1-496-9238: Fax: [phone]
Tu.A.26 I
THE DIHYDROPYRONES:THIRD-GENERATION, NON-PEPTIDIC HIV PROTEASE
INHIBITOR DEVELOPMENT CANDIDATES
Thaisrivongs, Suvit, Aristoff PATarpley WG, Chong KT, Watenpaugh KDTomich PK, Dolak
LA, Padbury GE, Schwende FJ, Zhao Z, Zipp GL, Kakuk TJ, Howe WJ, Romines KR, Romero
DL, Chrusciel RA, Gammill RB. Pharmacia & Upjohn, Inc., Kalamazoo, MI USA
Objective: Having introduced two generations of orally bioavailable, non peptidic HIV protease inhibitors: U-96988 (in 1993) and U- 1030 17 (in 1994) into phase I clinical trials, the
aim was to optimize third-generation non-peptidic inhibitors with good PK property and
oral broavailability and with significant improvement in antiviral potency especially in the
presence of plasma proteins.
Methods: Crystal structures of a series inhibitors complexed with the HIV protease assisted
the structure-based design effort. Analogues were assayed for inhibitory activity against
purifed HIV protease. Antiviral activity was assessed versus multiple HIV- I laboratory and
clinical isolates in several cell types. For PK studies, compounds were administered iv and
orally to rats and dogs.
Results: The third-generation non peptidic compounds were identified in the dihydropyrone
class: potent inhibitor of HIV protease (Ki < 0.05 nM); high antiviral activity (IC50 = 50 nM
in HIV-I IIIB infected H9 or MT4 cells; IC50 = 30 nM in HIV -IJRCSF infected PBMC; and
IC90 (median) - 0.3 pM against a panel of ten AZTresistant clinical HIV isolates in PBMC).
In an assay medium with added 75% hum an plasma, there was an approximately half -a-logunit increase in the IC50 value. After oral administration to dogs (Cmax - 26 pM at 10
mg/kg), the bioavailability was 40%.
Conclusion: Orally bioavaiable third generation, non-peptidic HIV protease inhibitors in the
dihydropyrone class have been optimized with in vitro antiviral potency comparable to the
peptide-derived inhibitors (saquinavir, indinavir, ritonavir).Very significantly studies revealed
that HIVI isolates highly resistant to ritonavir and broadly cross-resistant to peptide-derived
inhibitors (saqu inavir, indina vi, AG 1343) remained sensitive to these dihydropyrone
inhibitors. Extensive preclinica studies are underway in order to initiate clinical trials in the
second half of 1996.
SuvitThaisrivongs, 7255-209-0 19, Pharmacia & Upjohn, Inc., 301 Henrietta Street,
Kalamazoo, MI 4900 1-0199 Telephone: [phone], Fax: [phone],
email: sthaisri@cpwinect. upj.comi

Bi lione, Mimn, Alsin,.P2000 Rosario, Sta. Fe, Argentina.Tel.:387236 -
Tu.A. 165
EVIDENCE FOR THE HTLV-Ila AND HTLV-Ilb SUBTYPES IN SOUTHERN EUROPE.
Vallejo A1,2, Ferrante P3. Soriano V2, Calabr ML4, Mancuso M5, Heredia A1, Mannella F6,
arc a Siz A2, GonzalezLiho a2, Hewlett1. FDA/CBER, Bethesda, MD, n.S.A.2 Inst.
Salud atrlos III, Madrid, Spain. 3Univ. Milan, Italy. 4Univ. of Padova, Italy.5Don C. Gnocchi
Found. Milan, Italy.t talian Red. Cross, Rome, Italy.
human n Tcell ly photropc virus typ e (HTLV II) has been subtyped into two major
groups, Iia and lib, according to molecular studies involving env gene sequencing.
Subsequ ently, this retrovirus was fur ther sub classifi ed by examining the long terminal repeat
(LTR),the most divergent enornic region.Sequence analysis and restriction fiagment length
polymorphism (RFLP) applied to the LTR region identified either four or five groups within the
Ila subtype (depending on the restriction enzyme sets used) and six within the lib subtype.
In this study we analyzed the ILTR sequences of 29 samples obtained from HTLV II-infected
individuals living in Spain and Italy which included 24 injecting drug users (IDUs), 3 blood
donors and 2 subjects at nsk for HfIV/HTLV infection. Sequence analysis and phylogenetic