Design and synthesis of αGal-conjugated peptide T20 as novel antiviral agent for HIV-immunotargeting

Article abstract of DOI:10.1039/b313844e

An efficient chemo-enzymatic synthesis of α-Gal-conjugated peptide T20 as novel HIV-immuno-targeting agent is described. The synthesis involves chemo-enzymatic preparation of maleimide-functionalized αGal epitope and its chemoselective ligation with the peptide T20. The title compound contains two functional domains: the trisaccharide αGal epitope that binds to human natural anti-Gal antibodies and the 36-amino acid gp41 peptide (T20) that recognizes the gp41 N-terminal ectodomain of the HIV envelope. Biological assays demonstrated that the synthetic conjugate could readily bind to natural anti-Gal antibodies (both IgG and IgM type) in normal human serum and exhibited potent anti-HIV activity even in the absence of human antibodies and complement system. The experimental data suggest that the synthetic αGal-T20 might be valuable for in vivo HIV-immuno-targeting via antibody-mediated cytotoxicity and/or antibody-dependent, complement-mediated lysis of HIV particles and HIV-infected cells, thus providing an additional dimension of HIV intervention.

Full text of DOI:10.1039/b313844e

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Design and synthesis of ꢀGal-conjugated peptide T20 as novel
antiviral agent for HIV-immunotargeting
Kannan P. Naicker, Hengguang Li, Alonso Heredia, Haijing Song and Lai-Xi Wang*
Institute of Human Virology, University of Maryland Biotechnology Institute,
University of Maryland, Baltimore, MD 21201, USA
Received 31st October 2003, Accepted 12th January 2004
First published as an Advance Article on the web 2nd February 2004
An efficient chemo-enzymatic synthesis of αGal-conjugated peptide T20 as novel HIV-immuno-targeting agent is
described. The synthesis involves chemo-enzymatic preparation of maleimide-functionalized αGal epitope and its
chemoselective ligation with the peptide T20. The title compound contains two functional domains: the trisaccharide
αGal epitope that binds to human natural anti-Gal antibodies and the 36-amino acid gp41 peptide (T20) that
recognizes the gp41 N-terminal ectodomain of the HIV envelope. Biological assays demonstrated that the synthetic
conjugate could readily bind to natural anti-Gal antibodies (both IgG and IgM type) in normal human serum
and exhibited potent anti-HIV activity even in the absence of human antibodies and complement system.
The experimental data suggest that the synthetic αGal-T20 might be valuable for in vivo HIV-immuno-targeting
via antibody-mediated cytotoxicity and/or antibody-dependent, complement-mediated lysis of HIV particles and
HIV-infected cells, thus providing an additional dimension of HIV intervention.
known that natural anti-Gal antibodies are abundant in
humans, which consist of 1–2% total IgG and 3–8% total IgM
Introduction
The human immunodeficiency virus (HIV) is the cause of
AIDS.1–3 The worldwide pandemic of HIV/AIDS urges devel-
opment of more effective anti-HIV therapy.4 Hitherto, clinically
available anti-HIV drugs belong to two classes of anti-retroviral
agents: the reverse transcriptase inhibitor or protease inhibitor.5
The highly active antiretroviral therapy (HAART) that com-
bines two or more reverse transcriptase/protease inhibitors has
prolonged the survival of AIDS patients and significantly
reduced viral load of HIV infection. However, the toxicity and
emergence of drug-resistant HIV strains associated with these
anti-HIV drugs have limited their effectiveness. Recently, a
novel 36-amino acid gp41 peptide, T20, was approved by the
US Food and Drug Administration (FDA) as a new class of
anti-HIV drug. Peptide T20 is a potent HIV-cell fusion inhibi-
tor that exerts its anti-HIV activity through binding to the
fusion-essential N-terminal region of HIV envelope glyco-
protein gp41.6,7
in human serum.8 The interaction between human natural anti-
Gal antibodies and the αGal epitopes (oligosaccharides with
terminal Galα1–3Gal structure) on cells of most mammals such
as pigs is mainly responsible for the hyperacute rejection in
xenotransplantation.9–11 Major αGal epitopes that are abund-
antly expressed on cells of most mammals, except humans,
apes, and other Old World monkeys, are Galα1-3Galβ1-4-
Glcβ1-R, Galα1-3Galβ1-4GlcNAcβ1-R, and Galα1-3Galβ1-
4GlcNAcβ1-3Galβ1-4Glcβ1-R. We hypothesize that tagging
HIV surfaces with αGal epitopes will lead to antibody-
mediated cytotoxicity and/or antibody-dependent, comple-
ment-mediated lysis of HIV particles and HIV-infected cells. To
test the hypothesis, we have designed and synthesized novel
glycoconjugates that contain two functional domains, the
αGal epitope that binds to human natural antibodies and the
HIV-recognizing domain that binds to the HIV envelope. We
reasoned that the bi-functional glycoconjugates as mediators
will be able to direct human anti-Gal antibodies to HIV and
lead to subsequent complement cascade immuno-reactions
(Fig. 1). In this paper, we report the design and synthesis of the
To explore new approaches for anti-HIV therapy, we initiated
a project aimed at combining the power of antiviral agents and
intrinsic human natural antibodies to block HIV infection. It is
Fig. 1 Schematic description of αGal-mediated antibody coating of HIV surface.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 6 6 0 – 6 6 4
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
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Scheme 1 Synthesis of maleimide-functionalized aGal trisaccharide.
αGal-peptide T20 conjugate. The anti-HIV activity of the
readily converted the chloride into the azide 3 (80%). De-O-
synthetic conjugate and its binding to anti-Gal antibodies in
human serum are also described.
benzoylation of 3 with MeONa–MeOH gave the free lactoside
4 in quantitative yield. Enzymatic transfer of a galactose
moiety from uridine 5Ј-diphosphate galactose (UDP-Gal) to
the 3Ј-position of the lactoside 4 under the catalysis of the
recombinant α1-3-galactosyltransferase afforded the desired
trisaccharide derivative 5 in 94% yield. The α-configuration
of the newly formed glycosidic linkage was confirmed by the
relatively small coupling constant of the H-1Љ anomeric proton
(J1Љ,2Љ = 3.6 Hz). The azido group in 5 was then reduced through
catalytic hydrogenation to give the amino compound 6. Finally,
reaction of amine 6 with N-[β-maleimidopropyloxy] succin-
imide ester in an aqueous buffer gave the maleimide-functional-
ized αGal trisaccharide 7 in 76% yield, which was purified by
gel filtration and HPLC and characterized by ESI-MS.
Results and discussion
Synthesis of maleimide-functionalized ꢀGal epitope
For the conjugation of the αGal epitope to peptide or other
HIV-binding agents, a functionalized αGal epitope that would
allow chemo-selective ligation in a later stage would be desir-
able. Previously, we have applied the maleimide–thiol reaction
as a highly chemoselective ligation method for constructing
large and complex bio-conjugates in aqueous solution.12–14 Here
we adopted the same strategy for synthesizing the title oligo-
saccharide–peptide conjugate via a maleimide-functionalized
αGal epitope. Several chemical and chemoenzymatic methods
have already been reported for the synthesis of αGal epitope
oligosaccharides and some derivatives.15–22 Wang et al. carried
out pioneering work in the field and synthesized various αGal
derivatives for biological studies.23,24 Trisaccharide Galα1-
3Galβ1-4Glcβ1-R is one of the major natural αGal epitopes.
We used a chemo-enzymatic approach to synthesize the male-
imide-functionalized trisaccharide using a recombinant α1-3-
galactosyltransferase as the enzyme.18 The synthesis is summar-
ized in Scheme 1. To install a maleimide functionality in the
αGal epitope through a hydrophilic linker, per-O-benzoyl lacto-
syl bromide (1)25 was reacted with 2-(2-chloroethoxy)ethanol
under the catalysis of HgO/HgBr2 to give the β-lactoside deriv-
ative 2 in 78% yield. The desired β-anomeric configuration was
confirmed by the relatively large coupling constant of the ano-
meric proton (J1,2 = 7.8 Hz). Treatment of 2 with NaN3 in DMF
Synthesis of the ꢀGal-conjugated T20
T20 is a potent HIV entry inhibitor consisting of a 36-amino
acid sequence derived from the C-terminal ectodomain of
HIV-1 gp41.6,7 We synthesized cysteine (Cys)-tagged T20
with the cysteine residue being attached at either the N- or
C-terminus of the 36-amino acid peptide. Our cell fusion
experiments indicated that while the N-terminal tagged T20
(Cys-T20) maintained the same activity as T20 in inhibiting
HIV-cell fusion, the activity of C-terminal modified peptide
(T20-Cys) showed significantly reduced inhibitory activity
(data not shown). Accordingly, we have chosen the N-terminus
of T20 as the site for selective modification with αGal epitopes.
The chemoselective ligation of peptide Cys-T20 and the male-
imide-containing trisaccharide 7 proceeded efficiently in a
mixed solvent (MeCN–phosphate buffer, pH 7.2) to give the
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saccharide–peptide conjugate αGal-T20 (Scheme 2). The
product was purified by HPLC and its identity was character-
ized by ESI-MS (Fig. 2).
change its ability to bind to the gp41 region of the HIV
envelope.
To evaluate whether the synthetic αGal-T20 is able to recog-
nize anti-Gal antibodies in human serum, we performed a
simple enzyme-linked immunosorbent assay (ELISA). Mouse
laminin, a natural glycoprotein containing multiple αGal
epitopes, was included as a positive control in ELISA. αGal-
T20 or laminin was immobilized on a microplate and titrated
against serial dilutions of normal human serum (male, type
AB) that contains natural anti-Gal antibodies. Bound anti-
αGal IgG was detected by horseradish peroxidase (HRP)
conjugated anti-human IgG antibodies with subsequent color
reaction. It was observed that, similar to natural αGal-contain-
ing glycoprotein laminin, the synthetic αGal-T20 readily recog-
nized anti-αGal IgG in human serum (Fig. 4). Similar results
were observed for anti-αGal IgM in human serum when the
bound antibody was detected using HRP conjugated anti-
human IgM antibody in ELISA (data not shown). Although it
is difficult to provide a quantitative comparison of αGal-T20
and laminin, the data clearly indicate that the synthetic conju-
gate recognizes both anti-αGal IgG and IgM antibodies in
normal human serum. Taken together with the in vitro anti-
HIV activity of αGal-T20, our data suggest that the synthetic
αGal-conjugated T20 may act as a novel bi-functional antiviral
agent for synergistic HIV-inhibition and antibody-mediated
immuno-targeting of HIV in vivo. Therefore, the logical next
step of the research is to test the αGal-T20 and additional
synthetic αGal-conjugates in animal models to evaluate their
in vivo anti-HIV activities.
Scheme 2 Synthesis of aGal-conjugated peptide T20.
Fig. 2 The HPLC and ESI-MS profiles of synthetic αGal-T20.
Anti-HIV activity and antibody-binding ability of ꢀGal-T20
Fig. 4 Anti-Gal antibody binding to immobilized αGal epitopes. Data
from the average of two independent experiments were presented.
Symbols: ᭹, αGal-T20, ᭺, laminin.
The synthetic conjugate αGal-T20 was tested for its anti-HIV
activity in cell cultures. Peripheral blood mononuclear cells
(PBMCs) were used as host cells and HIV-1IIIB strain was used
as the virus for infection. Inhibition of viral infection was
assessed by measuring the viral reverse transcriptase (RT) activ-
ity. As demonstrated in Fig. 3, the synthetic αGal-T20 showed
almost the same inhibitory activity against HIV infection as
T20 in the absence of human antibodies. The measured IC50
(concentration for 50% inhibition) were 8 and 6 nM for αGal-
T20 and T20, respectively. The results suggest that attachment
of the trisaccharide epitope at the N-terminus of T20 does not
Conclusion
We have developed an efficient chemo-enzymatic synthesis of
αGal-conjugated peptide T20. The synthetic approach des-
cribed should be readily applicable for the synthesis of other
designed αGal-conjugates. Our preliminary studies indicate
that the synthetic αGal-T20 recognizes natural anti-αGal anti-
bodies in normal human serum and meanwhile exhibits potent
anti-HIV activity even in the absence of antibodies and com-
plementary systems. Our in vitro experimental data suggest that
the synthetic αGal-T20 would be valuable for in vivo immuno-
targeting against HIV via antibody-mediated cytotoxicity and/
or antibody-dependent, complement-mediated lysis of HIV
particles and HIV-infected cells, thus providing an additional
dimension of HIV intervention.
Experimental
General
1H NMR spectra were recorded on a Varian 500 with Me4Si
(δ 0) as the internal standard. ESI-MS spectra were measured
on a Waters ZMD mass spectrometer. Analytical TLC was
performed on glass plates coated with silica gel 60 F254
Fig. 3 Anti-HIV activity. Data from the average of two independent
experiments were presented. Symbols:᭹, T20; ᭺, αGal-T20.
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(E. Merck). Carbohydrates were detected by charring with 10%
sulfuric acid in ethanol, and amine was detected by ninhydrin
spraying (0.3% in ethanol and acetic acid (97 : 3)). Flash
column chromatography was performed with silica gel 60 (EM
Science, 230–400 mesh). Analytical HPLC was carried out with
a Waters 626 HPLC instrument on a Waters Nova-Pak C18
column (3.9 × 150 mm) at 40 ЊC. The column was eluted with a
suitable gradient of MeCN–H2O containing 0.1% TFA at a
flow rate of 1 mL minϪ1. Preparative HPLC was performed
with a Waters 600 HPLC instrument of a Waters C18 column
(Symmetry 300, 19 × 300 mm). The column was eluted with a
suitable gradient of MeCN–H2O containing 0.1% TFA at a
flow rate of 12 mL minϪ1. Mouse laminin, human serum (male,
type AB), peroxidase-conjugated goat anti-human IgG and
IgM, and TMB were purchased from Sigma. Recombinant
α1-3-galactosyltransferase was a gift from Dr Peng George
Wang. All other reagents and solvents were purchased from
Aldrich/Sigma.
selected 1H NMR data (500 MHz, D2O): δ 4.55 (d, J = 7.68 Hz,
1H, H-1), 4.47 (d, J = 8.05 Hz, 1H, H-1Ј), 3.55–4.11 (m, 17H),
3.54 (t, 2H, CH2N3), 3.36 (t, 1H); ESI-MS: 456.36 (M ϩ H)ϩ,
478.31 (M ϩ Na)ϩ.
2-(2-Azidoethoxy)ethyl
ꢀ-D-galactopyranosyl-(1 3)-ꢁ-D-
galactopyranosyl-(1 4)-ꢁ-D-glucopyranoside (5). A solution of
compound 4 (11 mg, 24.2 µmol) and UDP-Gal (20 mg, 32.7
µmol) in a Tris-HCl buffer (2 mL, 50 mM, pH 7.0) contain-
ing bovine serum albumin (BSA) (0.1%), MnCl2 (3.75 mg, 5.1
mmol), and recombinant α1-3-galactosyltransferase (0.4 unit)
was gently shaken at rt After 48 h, TLC (2-propanol–EtOAc–
water, 2 : 2 : 1, v/v) showed the disappearance of the di-
saccharide acceptor and the formation of a slower moving
product. The mixture was passed through a column of anion-
exchanging resin (Dowex-1, chloride form). The eluate was
concentrated and loaded onto a column of Sephadex G-10
using water as the eluent. The fractions containing the tri-
saccharride product were collected and lyophilized to give the
trisaccharide 5 (14 mg, 94%): selected 1H NMR data (500 MHz,
D2O): δ 5.1 (d, J = 3.6 Hz, 1H), 4.48 (d, J = 7.32 Hz, 1H, H-1),
4.46 (d, J = 7.32 Hz, 1H, H-1Ј), 4.14 (m, 2H), 3.50–4.04
(m, 21H), 3.47 (t, 2H, CH2N3), 3.30 (t, 1H); ESI-MS: 640.30
(M ϩ Na)ϩ, 618.29 (M ϩ H)ϩ, 489.06 (M Ϫ O(CH2)2-
O(CH2)2N3 ϩ H)ϩ.
2-(2-Chloroethoxy)ethyl 2,3,4,6-tetra-O-benzoyl-ꢁ-D-galacto-
pyranosyl-(1 4)-2,3,6-tri-O-benzoyl-ꢁ-D-glucopyranoside (2).
A mixture of hepta-O-benzoyl-α--lactosyl bromide25 (1.93 g,
1.7 mmol) and 2-(2-chloroethoxy)ethanol (1.1 g, 8.5 mmol) in
dry CHCl3 (20 mL) containing powdered molecular sieves (MS
4 Å, 3 g) were stirred at rt for 30 min. To the suspension were
added HgO (600 mg, 2.8 mmol) and HgBr2 (cat.) and the
resulting mixture was stirred in the dark at rt for 50 h. After
filtration through a pad of Celite, the filtrate was washed with
saturated NaHCO3 and water. The organic layer was dried over
anhydrous Na2SO4 and concentrated under reduced pressure.
The residue was subject to silica gel column chromatography
with hexane–EtOAc (3 : 2, v/v) as the eluent to afford 2 (1.5 g,
75%): 1H NMR (500 MHz, CDCl3): δ 8.1–7.1 (m, 35H), 5.83 (t,
J = 9.15 Hz, 1H, H-3), 5.73–5.77 (m, 2H, H-2Ј, H-4Ј), 5.49 (dd,
J = 9.52, 11.58 Hz, 1H, H-2), 5.41 (dd, J = 10.25 Hz, 1H, H-3Ј),
4.92 (d, J1Ј, = 8.05 Hz, 1H, H-1Ј), 4.83 (d, J1, = 7.8 Hz, 1H,
2-(2-Aminoethoxy)-ethyl ꢀ-D-galactopyranosyl-(1 3)-ꢁ-D-
galactopyranosyl-(1 4)-ꢁ-D-glucopyranoside (6). A solution of
azide 5 (12 mg, 20.28 µmol) in MeOH (10 mL) containing Pd/C
(5%, 3 mg) was stirred under an H2 atmosphere at rt over-
night. The solution was filtered through a pad of Celite. The
filtrate was concentrated under reduced pressure. The residue
was dissolved in water and lyophilized to give the amine 6
(11 mg, 92%). Selected 1H NMR data (500 MHz, D2O): δ 5.18
(d, J = 3.6 Hz, 1H), 4.56 (d, J = 6.58 Hz, 1H, H-1), 4.55 (d,
J = 7.69 Hz, 1H, H-1Ј), 4.22 (m, 2H), 3.56–4.12 (m, 24H), 3.38
(t, 1H); ESI-MS: 592.53 (M ϩ H)ϩ.
2Ј
2
H-1), 4.56–4.45 (m, 2H, H-6a,b), 4.29 (t, J = 9.51 Hz, 1H, H-4),
3.96–3.83 (m, 4H, H-5,5Ј, 6Јa, b), 3.78–3.53 (m, 6H), 3.49 (t,
3H); ESI-MS: 1177.80 (M ϩ H)ϩ, 1199.80 (M ϩ Na)ϩ, 1053.71
(M Ϫ O(CH2)2–O(CH2)2Cl ϩ H)ϩ.
2-(2-ꢁ-Maleimidopropylamidoethoxy)ethyl
ꢀ-D-galacto-
pyranosyl-(1 3)-ꢁ-D-galactopyranosyl-(1 4)-ꢁ-D-gluco-
pyranoside (7). To a solution of amine 6 (32 mg, 61.6 µmol) in a
phosphate buffer (5 mL, 50 mM, pH 7.2) was added a solution
of N-(β-maleimidopropyloxy)succinimide ester (25 mg, 96
µmol) in MeCN (3 mL). The mixture was stirred at rt for 3 h
when TLC showed the disapperance of the amine 6. The reac-
tion mixture was lyophilized and the product was then purified
by HPLC (0–30% MeCN) to give compound 7 (35 mg, 76%).
Selected 1H NMR data (500 MHz, D2O): δ 6.76 (s, 2H), 5.08 (d,
J = 3.6 Hz, 1H), 4.46 (d, J = 8.50 Hz, 1H, H-1), 4.45 (d, J = 8.50
Hz, 1H, H-1Ј), 4.13 (m, 2H), 3.58–3.98 (m, 24H), 2.54 (t, 2H),
2.39 (t, 2H); ESI-MS: 743.44 (M ϩ H)ϩ, 765.41 (M ϩ Na)ϩ.
2-(2-Azidoethoxy)ethyl 2,3,4,6-tetra-O-benzoyl-ꢁ-D-galacto-
pyranosyl-(1 4)-2,3,6-tri-O-benzoyl-ꢁ-D-glucopyranoside (3).
To a solution of chloride 2 (1.5 g, 1.45 mmol) in DMF (35 mL)
was added NaN3 (250 mg, 3.8 mmol). The resulting mixture
was stirred at 80 ЊC for 7 h. The solvent was removed under
reduced pressure. The residue was dissolved in CHCl3 (60 mL)
and washed with brine and water. The organic layer was dried
over anhydrous Na2SO4 and the solvent was removed under
reduced pressure. The residue was subject to silica gel chrom-
atography using hexane–EtOAc (3 : 2, v/v) as the eluent to give
1
azide 3 (1.37 g, 80%): H NMR (500 MHz, CDCl3): δ 8.1–7.1
(m, 35H), 5.82 (t, J = 9.15 Hz, 1H, H-3), 5.74–5.71 (m,
2H, H-2Ј, H-4Ј), 5.47 (dd, J = 9.51, 8.78, Hz, 1H, H-2), 5.39
Chemoselective ligation of peptide Cys-T20 and maleimide 7.
Peptide Cys-T20 was synthesized on a Pioneer solid phase
peptide synthesizer using Fmoc chemistry and was purified by
(dd, J = 10.62, 10.25 Hz, 1H, H-3Ј), 4.90 (d, J1Ј, = 7.68 Hz,
2Ј
1H, H-1Ј), 4.80 (d, J1, = 8.05 Hz, 1H, H-1), 4.65–4.45
HPLC according to our previously described procedure.12,14
A
2
(m, 2H, H-6a,b), 4.26 (t, J = 9.51 Hz, 1H, H-4), 3.96–3.82
(m, 4H, H-5,5Ј, 6Јa, b), 3.78–3.53 (m, 6H), 3.41 (t, 3H);
ESI-MS: 1184.42 (M ϩ H)ϩ, 1206.46 (M ϩ Na)ϩ, 1053.39
(M Ϫ O(CH2)2–O(CH2)2N3 ϩ H)ϩ.
mixture of Cys-T20 (9 mg, 1.95 µmol) and maleimide 7 (1.45
mg, 1.95 µmol) in a mixed solvent of phosphate buffer (pH 7.2,
50 mM, and 2.5 mL) and MeCN (1.5 mL) was gently shaken at
rt for 3 h. The reaction mixture was then lyophilized and the
ligation product was purified by preparative HPLC to give
αGal-T20 (8.4 mg, 81%). Analytical HPLC, tR = 17.3 min
(0–70% MeCN containing 0.1% TFA in 20 min); ESI-MS
1780.41 (M ϩ 3H)3ϩ, 1335.59 (M ϩ 4H)4ϩ, 1068.82 (M ϩ
5H)5ϩ.
2-(2-Azidoethoxy)ethyl-ꢁ-D-galactopyranosyl-(1 4)-ꢁ-D-
glucopyranoside (4). To a solution of O-benzoylated lacto-
side 3 (60 mg, 51 mmol) in dry MeOH (10 mL) was added a
catalytic amount of MeONa in MeOH. The solution was
stirred at rt overnight and then neutralized with cation-
exchange resin (Dowex 50WX8, Hϩ form). After filtration, the
filtrate was concentrated to dryness under reduced pressure.
The residue was crystallized from MeOH–EtOAc to give com-
pound 4 as white crystals (23 mg, 98%): mp 142.5–143.5 ЊC;
Anti-viral assays. Peripheral blood mononuclear cells
(PBMCs) were separated from whole blood of HIV-
seronegative donors by density centrifugation over Ficoll-
Hypaque (Sigma). The culture medium consisted of RPMI
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supplemented with 10% fetal bovine serum (FBS), 2 mM glu-
tamine and penicillin/streptomycin (Gibco, Grand Island, NY).
For infection studies PBMCs were stimulated with 2.5 µg mlϪ1
phytohemagglutinin (PHA; Boehringer Mannheim, India-
napolis, IN) for 3 days. Stimulated PBMCs were infected by
incubation with HIV-1IIIB at a multiplicity of infection of 1000
TCID50/106 PBMC for 2 h. PBMCs were then washed three
times with PBS and cultured at 37 ЊC in RPMI/10% FBS sup-
plemented with 100 units mlϪ1 rIL-2 (Boehringer Mannheim)
and the antiviral agents (αGal-T20 or T20). PBMCs were
seeded in 96-well flat-bottom plates at a density of 2 × 105
PBMCs per 200 µl. Following 3 days of culture, half of the
medium was replaced with fresh medium containing IL-2 and
the antiviral agents (αGal-T20 or T20). After 7 days of culture,
virus production in the culture supernatant was assayed by
measuring HIV-1 reverse transcriptase activity as described.26
Cell viability in culture in the presence or absence of antiviral
agents was measured by commercial MTT assay, as per the
manufacturer’s instructions (Boehringer Mannheim).
Biotechnology Institute, and the National Institutes of Health
(NIH grant AI54354 to LXW).
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We thank Dr Peng George Wang for kindly providing the
recombinant α1-3-galactosyltransferase. We also thank Dr
Robert Powell for assistance in ELISAs and Mr Nhut Le for
assistance in anti-viral assay. The work was partly supported by
the Institute of Human Virology, University of Maryland
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 6 6 0 – 6 6 4
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