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

Mo.A. 1071 - Mo.A.1075

Monday, July 8, 1996

Methods: The proteins investigated were lactoferrin, u-lactalbumin, r-lactoglobulirn A and flactoglobulin B.The capability to inhibit viral infectivity was assessed in a MT4 cell test system. Peptide mapping was performed to study the interaiction of the rmodlied proters with
the env pr.iten.
Results: Of milk proteins tested only native lactoferrin originated from bovine milk,
bovine colostrum. human milk and human plasma was able to completely inhibit HIV I
replication.The ri tro IC50 values ranged between 500 and 1500 nM.The other native
proteins had no effect. By acylation of the amino function of the amino acid lysin in the protein using anhydrides of succinic acid or cis-aconitic acid, proteins were synthesized that all
showed a strong antiviral activity against HIV type I and/or II.The in vitro IC50 values of
aconitylated proteins were in concentration range of 0.3 to 3 nM. Peptide scanning studies
indicated that the native lactoferrin as well as the charged modified proteins strongly bind
to theV3 loop of the HIV-I gpl20 envelope protein in the same concentration range as
above mentioned IC50
Conclusions: The underlying mechanism is likely that by shielding of the V3-domain of
gpI 20 this results in inhibition of the cellular fusion and entry of the virus in MT4 cells.
Succiny ationr aconitylation of (x-lactalbumin and B-lactoglobulin A/B also produced strong
ant HI V2 acwity with IC50 values in the order of 500 to 3000 nM. AII compounds
showed virtuilly no cvtotoxicity in the concentration range used.
dr. Has (Gi Huisrni, Central Laboratory of the Netherlands Red Cross Blood Transfusion
Service. Dept Developrmental Reseasrch, Plesmanlaan 125, 1066CX Amrrsterdarn,The
Nether-lands. tel.: 20-51l23124, fax: [phone].
Mo.A. 107 I1
INHIBITON OF ACUTE HIV- I INFECTION OF CD4+ T CELLS BY LITHIUM
GAMOLENATE
Randall, ShaIron, Win ther, M. D.*, Horrobin, D. F.*, Chan. W.L. Dept. offVirology, St.
Bartholomew' Hospital Medical Coliege, London, UK: *Scotia Pharmaceuticals Ltd.,
l'.Iova Scotia, C1 rnada.
Objectives: 1 he tatty acid metabolism ofT cells has been shown to be altered in HIV I,rfection. We are inv estigating the possible development of a new therapeutic approach to
HIV -I infectior, by targeting fatty acid metabolism in the host cell. Syncytium formation,
which plays an r por tant role in the mediation and spread of virus in HIV infection,
depends on the expression of CD4 and cell adhesion molecules on the surface of the host
cell.The aim of this study is to investigate the effect of the lithium salt of gamma linolenic
acid (LiGLA), anr essential fatty acid, on acute HIV- I infection of C8 I 66 lymphoblastoid cells
for (i) possible inhibition of virus infection, (ii) selective toxicity for HIV-infected cells and (iii)
how such effect(s) relate to the expression of CD4 and adhesion molecules on the surface
of the host cell.
Methods: Two-colour flow cytometric analysis was carried out using fluoroscein conjugated
anti p24 monoclonl antibody (MAb) to detect HIV infection of cells, in conjunction with
ethidium rmonoazide bromide to ascertain cell viability In addition, a non radioactive assay
was used to quantitate the amount of reverse transcriptase (RT) in cell and supernatant
samples and p24 antigen released into infected culture fluids. Flow cytometric analysis of fluoroscein -labelled MAbs against cell surface molecules CD4, integrins LFA-I alpha and beta,
LFA-3 and the intercellular adhesion molecules (ICAMs) were used to determine their
expression on LiGLA-treated cells.
Results: Using two -colour flow cytometry LiGLA was found to inhibit acute HIV I infection
of C8 I 66 cells in the absence of any significant difference in cell viability between infected
and uninfected cells. In addition, virus RT and p24 production were markedly reduced by
treatment with LiGLA.There was a concomitant decrease in cell surface expression of CD4,
LFA- I alpha ard beta and ICAM-2 with the reduced infectivity seen in LiGLA-treated cells.
Conclusions: These results suggest that down regulation of CD4, LFA- I and ICAM-2 may
be responsible for the reduced virus infectity demonstrated, as syncytium formation can
be affected by reduced cell adhesion.
S. L. Randall, Dept. of Virology St. Bartholomew's Hospital Medical College, 5 I - 53
Bartholomew Close, VVWest Smithfeld. London EC IA 7BE, UK.
Mo.A. 1072
ASSESSMENT OF ANTIRETROVIRAL THERAPY ON ACTIVATION-INDUCED CELL
DEATH IN HIV POSITIVE INDIVIDUALS
Johnson, Nicholas, Stone J., Pinching A.J., Parkin J.M. St. Bartholomews Hospital, London,
United Kingdom
Objectives: To assess the effect of antiretroviral therapy on inr vitro lymphocyte activationinduced cell death (AICD) in HIV infected patients. Compare this to changes in CD4 cell
numbers and assess AICD as a means of monitoring antiretroviral therapy
Methods: Eight patients were sampled before and after initiation of therapy. Four antiretroviral naive patients starting therapy (AZT and DDC or DDI), two recommencing
monotherapy after six months off treatment, and two adding a second antiretroviral to
AZT. AICD was measured within peripheral blood lymphocytes (PBMC) 72 hours following
stimulation with phytohaemagglutanin (PHA) by flow cytometry Results were compared
against standard laboratory disease markers including CD4 lymphocyte count, using
Wilcoxon signed-rank analysis.
Results: The m an fillow up for each patient was 12 weeks.Within this period the CD4
lymphocyte count rncreased fbom a mean value of 158 cells/mm3 to 198 cells/mm3.
However, this rid not prove to be significant (p-0.34). Over the same time interval AICD
decreased from a value of 33.8% (SD 1 16) to 6.97% (SD = 2.9).A decrease in AICD was
observed in eer y patient and reached significance (p=0.01).A similar decease in AICD was
observed within the CD4 lymphocyte population falling from 30.6% to 4.47% (p--0.07).
Conclusions: AICD declined significantly in patients undergoing antiretroviral therapy to levels expected fur HIV negative controls.This occurs over a time period of between 8 and I6
weeks, similar in time scale to improvements in lymphocyte function and intracellular signalling (Nye, K. E. et Il. AIDS 5: 41 I 3, I99 I), but much slower than changes in HIV viraemia.
This suggests that the decrease in AICD is related to the emergence of lymphocytes that
have not encountered virus or viral products.This data also indicates that AICD could be
used to follow the effectiveness of antiretroviral treatment.
N. Johnson, Dept. of Immunology St. Bartholomews Hospital, London, EC IA 7BE, UK.Tel.
01 7 I 60!8 04 Fax. [phone] email [email protected]

Mo.A. 1073
ASSESSMENT OF HIV INHIBITORY ACTIVITY IN SALIVA AND OTHER BODY
FLUIDS.
Kazmi, Sharnu.in MH, ul; jE, O'Shea, 5, Banatvala, JE,. Challacombe, SJ. Sweet, SP Oral AIDS
Research Centre,Dept. Oral Medicine and Pathology, Guys Hospital, London, UK.
Objectives: To perfcir' a comparative evaluation of the HIV inhibitory activity of a number
of body fluids ii rud ng saliva, breast milk and seminal plasma.This was done in parallel with
a gp I 20 binding plant lectin from Golanthus nivolis which was used as a positive control.
Methods: HIV inhibitory activity was evaluated by incubating body fluids or lectin for I hour
at 37~C with cell free virus from a laboratory adapted s-rain of HIV Then samples were
inoculated into C8 166 cell cultures. After 24 hours, cultures were washed and incubated for
a further 6 days. HIV replication was monitored by syncyt um formation and quantitation of
p24 antigen.
Results: The results indicated that, of the body fluids tested, whole and parotid salivas
demonstrated the highest inhibitory activity to HIV Submnandibular/sublingual saliva demonstrated less activity but we have noted that there is wide variation in inhibitory activity
between subjects. Seminal plasma and breast milk also showed considerable anti-HIV activi
tyThe plant lectin demonstrated high anti-HIV activity but at a much greater concentration
than normal physiological levels (I mg/ml).
Conclusions: The results suggest that there are a number of naturally occurring factors present in various types of body fluids that clearly demonstrate inhibitory activity to HIV.
Isolation and characterisation of these factors could lead to their potential use in a virucidal
gel formulation to minimise HIV transmission.
S Kazmi, Dept Oral Medicine, Floor 28, Guy's Hospital, London SE1 9RT UK.
Tel. [phone], Fax. [phone]
Mo.A. 1074
INHIBITION OF HIV REPLICATION BYTHE PLANT PHYLANTHUSAMARUS
Mullen ane E, I O'Shea S, Rostron T I Houghton PJ,2 Woldermariam TZ,2 Walker E,.3
Banatvala JE, Thyagarajan SP4 I Dept. ofVirology United Medical and Dental Schools of
Guys and St.Thomass Hospitals, London, UK; 2Dept. of Pharmacy, Kings college. London,
UK;3Scottish Centre for Infection and Environmental Health, Ruchill Hospital, Glasgow,
Scotland: 4Dept. of Microbiology University of Madras, India
Objective: Plants of the genus Phyllanthus are widely used by traditional medical practitioners for the treatment of jaundice and other diseases and are very well tolerated. Phyllanthus
onrrnarus (PA) inhibits the DNA polymerase of hepatitis B virus and chronic carriers treated
with the plant extract clear hepatitis B surface antigen. As PA has also been shown to inhibit
reverse transcriptase its effect on the in vitro replication of HIV has been evaluated.
Methods: The anti viral effect of a crude aqueous extract of PA and I 2 purified fractions
prepared using either polyamide, reverse phase silica and Sephadex G-25 columns (4 fractions per column) were evaluated.Titres of laboratory adapted (H9RF) and wild type isolates of HIV were determined in the presence and absence of PA by syncytium formation
in C81 66 cell cultures and quantitation of p24 antigen in culture supernatants using EIA.
Results: PA was non toxic to cell cultures and delayed the appearance of syncytia from 24
to 96 hours. HIV infected cultures treated with 250 pg/mil of crude PA had approximately a
100 fold lowerThe purified fractions when used individually or in combination decreased
the level of virus replication.The most active of the purified fr-actions suggested that the
active compounds are relatively non polar of high molecular mass and have some phenolic
properties.
Conclusions: PA can inhibit the in vitro replication of HIV and preliminary work has identi
fied potential active compounds. Further studies are therefore warranted to evaluate the
potential use of PA as a therapeutic agent in HIV infection.
J Mullen, London SE I 7EH, United Kingdom.Tel: [phone]x3 I127;
Fax: [phone]
Mo.A. 1075
DISCOVERY OF POTENT, ORALLY BIOAVAILABLE, NON-PEPTIDIC, CYCLIC
SULFONES AS HIV PROTEASE INHIBITORS
Choung U. Kim1, Larry McGee, Steve Krawczyk1. Erick Harwood,Yoko Harada. S.
SwaminathanT, Norbert Bischofberger1, Ming S. Chen I, Julie M. Cherrington I, Shely F.
Xiong, Andrew Mulato, Carmina Flores, Kenneth C. Candy, Linda Griffin, Reza Oliya I.
John W. Erickson2 I CUlead Sciences Inc., 353 Lakeside Drve, Foster City CA 94404,
-Structural Biochemistry Program, PRI/DynCorp, NCI-Fredrick Cancer, Research and
Development Center, Fredrick, MD 21702
A rational drug design strategy using crystallographic information generated from several
HIV Protease-inhibitor complexes has led to the discovery of a novel sen es of highly
potent, non-peptidic and cyclic inhibitors of the HIV protease.The new structure of these
compounds possesses a C2 symmetric diol which resembles the transition state of the
enzyme catalyzed reaction. In addition, the complete lack of the amide functionality in the
structure prevents these molecules from proteolytic cleavage.The structural water in the
active site was displaced by the oxygen atom of the SO2 group in the cyclic scaffold.The
side chains of the cyclic structure occupy only S I(S I) and S2($2) regions of the enzyme
active site.Those structural features are different from HIV protease inhibitors described
previously. One member of this class, GS 3333 (Ki < I nM, IC90 - 40 nM in MT2 cells) was
selected for further evaluation of pharmacokinetics and o al bioavailability. A single 10 mg/kg
oral dose of GS 3333 administered to dogs (n=5/group) gave the following pharmacokinet
ic profiles: F = 74%, Cmax - 3.3 pg/mL, ti/2 = 4.7 h.The duration that the plasma drug
concentration remained 40 times above IC90 exceeded 12 hours.This series of compounds
presented herein are potent inhibitors of HIV replication n vitro and exhibited excellent
pharmacological profiles.
Choung U. Kim, 353 Lakeside Drive, Foster City CA 94404 U.S.A.Telephone: 4 I5 572-66 I6
Fax: [phone]