Peptide sugar mimetics prevent HIV type 1 replication in peripheral blood mononuclear cells in the presence of HIV-positive antiserum.
ABSTRACT Cells of the immune system express a number of receptors that bind carbohydrate ligands. We questioned whether peptide mimetics of these ligands will activate phagocytic cells and thereby enhance an antiviral response. Short peptide sequences were identified by computational modeling of docking to glycan-specific lectins, selected as receptor analogs, and incorporated into quadravalent structures by peptide synthesis. A peptide with the sequence HPSLK bound to several lectins specific for monosaccharides and to lectins specific for Neu5Ac-Gal-containing complex glycans, whereas a longer sequence, NPSHPLSG, bound only lectins specific for the more complex glycans. In cultures of peripheral blood mononuclear cells (PBMCs) these peptides stimulated phagocytosis of opsonized microspheres. The peptides inhibited replication of HIV-1 in PBMC cultures by 20-80% at concentrations between 1 nM and 1 muM but inhibited replication 100% in the presence of diluted HIV-positive antiserum that alone inhibited replication by 30%. HPSLK caused about 50% loss of viability of cells at 1 mM, a concentration 10(6)-fold higher than an effective inhibitory concentration, but no toxicity was observed with NPSHPLSG. These results demonstrated that peptidomimetics of glycan ligands of cellular receptors are effective in activating phagocytosis, which may be a factor in providing complete inhibition of HIV-1 replication in vitro.
- SourceAvailable from: Laura EgginkGlycobiology Insights. 01/2010;
Peptide Sugar Mimetics Prevent HIV Type 1 Replication
in Peripheral Blood Mononuclear Cells in the Presence
of HIV-Positive Antiserum
Laura L. Eggink,1Maria Salas,2Carl V. Hanson,2and J. Kenneth Hoober1
peptide mimetics of these ligands will activate phagocytic cells and thereby enhance an antiviral response. Short
peptide sequences were identified by computational modeling of docking to glycan-specific lectins, selected as
receptor analogs, and incorporated into quadravalent structures by peptide synthesis. A peptide with the se-
quence HPSLK bound to several lectins specific for monosaccharides and to lectins specific for Neu5Ac-
Gal-containing complex glycans, whereas a longer sequence, NPSHPLSG, bound only lectins specific for the more
complex glycans. In cultures of peripheral blood mononuclear cells (PBMCs) these peptides stimulated phago-
cytosis of opsonized microspheres. The peptides inhibited replication of HIV-1 in PBMC cultures by 20–80% at
concentrations between 1 nM and 1mM but inhibited replication 100% in the presence of diluted HIV-positive
antiserum that alone inhibited replication by 30%. HPSLK caused about 50% loss of viability of cells at 1mM, a
concentration 106-fold higher than an effective inhibitory concentration, but no toxicity was observed with
NPSHPLSG. These results demonstrated that peptidomimetics of glycan ligands of cellular receptors are effective
in activating phagocytosis, which may be a factor in providing complete inhibition of HIV-1 replication in vitro.
viral content then gains access to the cytoplasm by fusion of
its envelope with the endosomal membrane. This pathway
attenuates the effectiveness of antibodies to eliminate the
virus through Fc-mediated phagocytosis. Although virion-
antibody complexes bind to Fc-receptor-bearing phagocytic
cells,3–9the activity of these cells becomes suppressed as
the infection progresses.3,10,11Elimination of the virus could
possibly be favorably manipulated by stimulation of the
Phagocytic cells are regulated by an extensive array of
cell surface receptors. Some are C-type lectins, which bind
sugars in a calcium-dependent manner.14,15A C-type N-
acetylgalactosamine (GalNAc)-binding receptor, CD301, is
expressed on the surface of macrophages and immature
dendritic cells and is involved in endocytosis.16,17C-type
lectins that also undergo endocytosis include DC-SIGN and
the mannose (Man) receptor, which bind Man; Langerin,
which binds galactose (Gal); and Dectin-1, a b-glucan recep-
IV-1, as other enveloped viruses,1initiates infection
by entering cells through an endocytic pathway.2The
tor.15Other receptors are the I-type lectins that belong to the
immunoglobulin superfamily. The best characterized mem-
bers of I-type lectins are siglecs (sialic acid-binding Ig-like
lectins), which bind sialic acid (5-acetylneuraminic acid,
Neu5Ac)-Gal-containing glycans and modulate signaling
events in the immune system.18,19Several siglecs, particularly
CD22 (siglec-2),20CD33 (siglec-3),21siglec-5,22and siaload-
hesin (CD169),18,19,23undergo endocytosis on binding of sia-
lylated ligands. Whereas most siglecs contain an inhibitory
immunoreceptor tyrosine-based inhibitory motif (ITIM) se-
quence in their cytoplasmic domain,18,19siglec-14 and siglec-
15 lack this domain and function in conjunction with an
activating adaptor protein, DAP10 or DAP12, which contain
Glycans have several drawbacks as therapeutic agents, in
particular, their difficult synthesis and stability. Conse-
quently, peptide mimetics of sugars are gaining interest as
substitutes.26We questioned whether short peptides would
mimic sugar components of natural ligands of receptors that
function to stimulate phagocytosis. Peptide mimetics of sug-
ars have potential advantages over glycans and glycoproteins
1Susavion Biosciences, Inc., Tempe, Arizona 85281.
2Viral and Rickettsial Disease Laboratory, California Department of Public Health, Richmond, California 94804.
AIDS RESEARCH AND HUMAN RETROVIRUSES
Volume 26, Number 2, 2010
ª Mary Ann Liebert, Inc.
because of chemical synthesis and ease of purification.
Moreover, peptide-based structures can be constructed that
bind with higher affinities to lectins than glycan ligands.27A
number of peptides that mimic sugars have been identified,
that act as more general mimetics.31Some peptides can
functionally mimic a sugar, such as those with the consensus
core sequence YPY that inhibit the mitogenic activity of the
Man-specific lectin concanavalin A (Con A), yet bind at a site
different from the saccharide-binding site.32,33Peptide mi-
metics have been studied as vaccines to elicit antibodies
against sugar antigens, including those on the surface of
HIV,27,31and complex oligosaccharides.34,35
We designed several short peptide sequences by compu-
tational modeling that were predicted to have significant
affinity to lectins that have the sugar specificity of several
receptors. These sequences were incorporated into multiva-
lent structures to achieve clustering of ligands and receptors,
features that are required for high-affinity interactions,19and
to accommodate the possibility that cross-linking of recep-
tors is required for activation of phagocytosis.36Well-
characterized plant lectins were used as receptor analogs to
analyze the mimetic properties of the peptides. In binding
assays, the peptide with sequence HPSLK had characteristics
of a general sugar mimetic and bound to several lectins with
higher affinity than their natural ligands. Highest affinities
were found with lectins that bind monosaccharides such as
Neu5Ac, GalNAc, or fucose. Longer peptides, in particular
NPSHPLSG, did not bind these lectins but bound strongly to
lectins specific for di- or trisaccharides that terminate with
Neu5Ac-Gal. Because these oligosaccharides are components
of complex glycan ligands that bind specific receptors, par-
ticularly siglecs, we determined the ability of the peptides to
activate phagocytosis of adherent cells in cultures of human
peripheral blood mononuclear cells (PBMCs). Nanomolar
of opsonized microspheres. The peptides had modest ability
was achieved in the presence of antibodies against the virus.
Methods and Materials
Peptide design and synthesis
Unique peptide sequences were designed by computer
modeling of docking to sugar-binding sites of lectins, down-
loaded from the Protein Data Bank, with ArgusLab 4.0.1
were synthesized on a trilysine core37,38utilizing Fmoc
(9-fluorenylmethoxycarbonyl)-protected amino acids and a
Milligen Biosearch 9050þcontinuous flow peptide synthe-
sizer (Millipore, Billerica, MA). The C-terminus consisted of
either an amide group (no tag), e-biotinyl-lysine, or b-alanine-
cysteine. A dansyl group was incorporated by reaction of
the thiol groupon C-terminal
(IAEDANS) (Molecular Probes Invitrogen, Eugene, OR). The
structure for the HPSLK peptide, with a C-terminal amide,
was [(HPSLKGGGS)2K]2K-NH2. The sequence GGGS was
included as a spacer to extend the active sequence from the
Peptides were also synthesized with the sequences
NPSHPSLG and NPSHPLSG. Peptides (200–300mg) were
dissolved in water, neutralized with Na2CO3, applied to a
column (1?5cm) of CM-Sephadex C-50, and washed exten-
sively with water to remove side-products of synthesis. Pep-
tides were eluted with 0.1N HCl, neutralized, and then
250mm) (Phenomenex, Torrance, CA) using a gradient of
acetonitrilein water containing 0.1% trifluoroacetic acid (TFA).
The eluted peptides were dried under vacuum, dissolved in
NaCl, and filter sterilized. Concentration was determined by
the bicinchoninic acid assay (Pierce, Rockland, IL) using the
dansylated peptide (extinction coefficient, emM¼5.7cm?1at
336nm) as standard. Correct synthesis was confirmed by
mass spectroscopy and amino acid sequence analysis.
For lectins available as peroxidase conjugates, 50ml of 2mM
biotin-tagged quadravalent peptide in phosphate-buffered
saline (PBS), pH 7.2, was added to each streptavidin-coated
well ofa microtiter plate (binding capacity, 125 pmol per well,
Pierce) and incubated 1h at room temperature. The wells
were blocked with 1% gelatin in 50mM Tris–HCl, pH 7.5, and
washed two times with 50mM Tris–HCl (pH 7.5) containing
150mM NaCl, 1mM CaCl2, 1mM MgCl2, and 1mM MnCl2
(buffer A). Then 50ml of 1mg=ml horseradish peroxidase-
conjugated lectins (Sigma-Aldrich) in buffer A were added.
After 1h incubation, wells were washed four times with
buffer A and then 50ml peroxidase substrate (1-Step Ultra
TMB-ELISA, Pierce) were added. After 2–5min the reaction
was stopped with 50ml 2M H2SO4and absorbance was read
immediately at 450nm. The amount of lectin bound was cal-
lectins (OD450=min=ng protein).
The protocol was modified to assay binding of peptides to
amurensis (MAA). Lectin-coated microwell strips (AlerCHEK,
Portland, ME) were hydrated in buffer A, blocked with 1%
gelatin in buffer A, and then 100 pmol of biotinylated peptide
were added to each well. After 1h incubation, the wells were
washed three times with buffer A and then 50ml of 0.3mg=ml
peroxidase-conjugated streptavidin (Sigma-Aldrich) were
added. Wells were washed four times with buffer A and
peroxidase activity was assayed as above.
Human PBMCs were purchased from Cellular Technology
Ltd. (Shaker Heights, OH) and cultured in RPMI-1640 me-
dium containing 10%fetalbovineserum (FBS).Dragon green-
labeled, streptavidin-coated microspheres (0.97mm diameter,
Bangs Laboratories, Inc., Fishers, IN) in 0.1% bovine serum
albumin and 0.05% Tween 20 were incubated with biotiny-
lated recombinant HIV-1 envelope gp41 (ProSpec-Tany
Technogene LTD, Rehovot, Israel), washed with PBS, opso-
nized withpolyclonal rabbit anti-gp41 (ProSpec), and washed
again with PBS. Alternatively, microspheres were incubated
with a 1:1 dilution of polyclonal rabbit antistreptavidin serum
(Sigma-Aldrich) and washed three times with PBS. PBMCs
150EGGINK ET AL.
were incubated 20h with 50 nM peptide in microtiter plates
and then microspheres were added at approximately a 10:1
ratio of microspheres to cells. After an hour of incubation,
formalin was added to a concentration of 2%, the samples
were allowed to stand at 48C overnight, and were then wa-
shed three times with PBS to remove free microspheres and
examined with an inverted microscope. Unfixed cells were
analyzed for phagocytosis of the fluorescent microspheres,
opsonized with the antistreptavidin serum, by flow cytome-
try with a 2100 Bioanalyzer (Agilent Technologies, Wald-
bronn, Germany). Cytochalasin D was used as an inhibitor of
phagocytosis. As the reference, SYTO?62, a red fluorescent
nucleic acid stain, was used and only doubly labeled cells
were counted as phagocytic.
Human PBMCs (Cellular Technology Ltd) were plated at
4?105cells per well in RPMI 1640 medium containing 10%
FBS. After 2 days at 378C in 5% CO2, peptide or lipopoly-
saccharide (LPS), as a positive control, at final concentrations
of 100 nM and 10ng=ml, respectively, was added and the
incubation continued for 4h. Media were then withdrawn
and cytokines assayed in duplicate with the 40-cytokine
human inflammation antibody array by RayBiotech, Inc.
HIV replication assays
HIV-1 replication was assayed in cultures of T (MT2) cells
Hanson at the California Department of Public Health, and
peptide at several concentrations between 0.1 nM and 1mM
were added to the culture and cells were immobilized under
agarose. After 6 days at 378C, the cells were stained with
propidium iodide and plaques were counted.
PBMCs were prepared through Ficoll gradients from buffy
coats purchased from the American Red Cross blood bank
under IRB approval held by the blood bank. Aliquots of
5–10?107cells were stored frozen in 90% FBSþ10% DMSO
in liquid N2. Cells were thawed, sedimented, suspended in
RPMI-1640 medium containing 25% FBS and 5% interleukin
(IL)-2 (ZeptoMetrix Corp. Buffalo, NY), and activated with
5mg=ml phytohemagglutinin (PHA) for 24h at 378C in 5%
CO2. Cells were washed free of PHA, suspended in RPMI-
a 96-well microtiter plate (50ml, 2.5?105cells=well). Peptide
(100ml) was added followed by 50ml of medium or diluted
HIV-positive serum, pooled from North American AIDS
patients (final dilution 1:360). R5 HIV-1 strain SF162 (clade B)
replication assayed according to a standard protocol.40,41SF-
162 was obtained through the NIH AIDS Research and
Reference Reagent Program from Jay Levy42and HIV-1
97ZA009 through the NIH AIDS Research and Reference
Reagent Program from Dr. Robert Bollinger and the UNAIDS
Network for HIV Isolation and Characterization. Cells were
incubated 3 days at 378C, then washed three times to remove
free virus, peptide, and antiserum and suspended to 250ml of
medium. After an additional 24-h incubation, cells were lysed
by ELISA to quantify the amount of virus.
Determination of cytotoxicity
Viability of cells was determined after staining with acri-
dine orange and ethidium bromide.43,44In this assay, viable
cells show green fluorescence whereas dead cells show red
fluorescence. Percent viability was determined by the number
of viable cells in peptide-treated cultures divided by viable
cells in cultures with vehicle alone.
Identification and synthesis of glycan mimetics
We previously reported45that the sequence VQATQ
SNQHTPR, identified during a screen of a phage display li-
brary with the lectin from Helix pomatia (HP), behaves as a
mimetic of Gal=GalNAc. Preliminary experiments indicated
that this peptide stimulated phagocytosis of bacterial cells
(L.L. Eggink and J.K. Hoober, unpublished data). To further
explore sugar mimetics as activators of phagocytes, addi-
tional, shorter peptides were designed by computational
modeling of docking to several lectins whose structures and
sugar-binding sites are well characterized. We initially chose
lectins specific for Gal, GalNAc, and Neu5Ac. HPSLK was
identified as an amino acid sequence predicted to bind these
lectins with a highly favorable binding energy.
with a solid-phase assay. The HPSLK peptide was synthe-
sized as monovalent, bivalent, or quadravalent constructs,
with the active sequence extended from a GGGS linker se-
quence attached to a mono-, di-, or trilysine core, respectively.
The C-terminus was extended with e-biotinyl-lysine to anchor
the peptide to streptavidin in wells of a microtiter plate. After
extensive washing, lectin retained in the wells was detected
by peroxidase activity conjugated to the lectin. As shown in
Fig. 1, strong binding to the qudravalent peptide was found
with lectins from Dolichos biflorus (DB), specific for GalNAc46;
Triticum vulgaris (wheat germ agglutinin, WGA), specific
for Neu5Ac, N-acetylglucosamine (GlcNAc),47,48and clusters
of GalNAc49,50; and Ulex europaeus (UEA1), specific for L-
fucose(a1-2)-D-Gal-containing oligosaccharides.51Less bind-
ing was obtained with lectins from Griffonia simplicifolia (GS),
specific for Gal,52and withHP, specific for GalNAcand Gal.53
A peptide in which the spacer was duplicated with the se-
quence VGGGS, as a control peptide, did not bind to these
We were particularly interested in whether multivalent
structures show enhanced binding to the lectins over a
monovalent peptide, as predicted by entropic factors54,55and
ligand density.31,56In an assay with equal numbers of sequences,
a bivalent structure bound to lectins about 50% as strongly
and the monovalent peptide about 10% as strongly as the
quadravalent peptide (Fig. 2). Quantitative competition
binding assays were performed to estimate affinity of the
HPSLK peptide to lectins. Neu5Ac (neutralized) was used as
competing monosaccharide with WGA, which binds to the
lectin with a Kdvalue of 1mM.57A 50% inhibition of binding
of the bivalent peptide was obtained at 90mM Neu5Ac and
22 nM was calculated from these data for binding of the biva-
lent molecule and of 7 nM for the quadravalent peptide to
WGA. Only 20% inhibition of binding of HPSLK to DB was
INHIBITION OF HIV-1 BY PEPTIDE SUGAR MIMETICS 151
obtained with 250mM GalNAc. To test for inhibition by more
complex glycans, porcine gastric mucin, which contains gly-
cans with GalNAc, Gal, GlcNAc, and fucose, with a minor
amount of Neu5A, as terminal sugars58,59was added. Mucin
(50mg=ml)inhibited bindingoftheHPSLKpeptidetoHP, DB,
competition experiments support the conclusion that the
Figure 1 shows that only a low amount of DB, WGA, and
GS remained bound to the NPSHPSLG peptide after the ex-
not detected with another quadravalent peptide containing
thesequence NPSHPLSG, inwhich theS andL positions were
exchanged. We questioned whether the longer peptides
would mimic more complex glycans. Lectin SNA1 from
Sambucus niger is highly specific for Neu5Ac(a2-6)Gal
and does not bind significantly to the monosaccharide,
Neu5Ac.60,61Binding to lectin MAA from Maackia amurensis
requires a trisaccharide and is specific for the Neu5Ac(a2-3)
Gal linkage.61,62As shown in Fig. 1, the two longer peptides,
as well as HPSLK, bound strongly to these two lectins. In
these assays, with the lectin bound to the solid phase, the
effect of concentration of peptide indicated that half-maximal
binding of HPSLK to SNA1 was obtained at 20 nM and
to MAA at 400 nM. In contrast, half-maximal binding of
NPSHPLSG to SNA1 was obtained at 200 nM and to MAA at
100 nM (data not shown). Thus, although the peptides bind to
both lectins, HPSLK bound with higher affinity to the lectin
specific for the a2-6 linkage whereas NPSHPLSG had greater
affinity for the lectin specific for the a2-3 linkage. These ex-
periments demonstrate that the sequence of the peptide, in
addition to valency, is critical for high affinity to these lectins.
As an additional control, peptides that lacked the biotin tag
were not retained in the assay. We chose HPSLK and
NPSHPLSG, based on data shown in Fig. 1,for further studies
on activation of phagocytic cells.
Activation of phagocytosis
We determined whether the peptides stimulate uptake of
opsonized beads.For these
and NPSHPLSG peptides were synthesized without the C-
terminal, e-biotinyl-lysine extension to avoid adventitious
binding of the biotin moiety. Antibody-mediated phagocy-
tosis was assayed by the uptake of streptavidin-coated mi-
crospheres to which biotinylated HIV-1 envelope gp41 was
bound and then opsonized with rabbit antibodies against
gp41. PBMC cultures were incubated 20h with 50 nM peptide
and then challenged with the microspheres. As shown in
Fig. 3A, an hour after adding the microspheres, cells in un-
treated cultures contained few if any beads. A relatively in-
active quadravalent peptide containing the sequence VSNQH
did not significantly stimulate uptake of beads over the
experiments, the HPSLK
purple, DB; blue, UEA1; orange, SNA1; light blue, MAA.
Binding of quadravalent peptides, containing the sequences shown, to lectins. Dark blue, HP; red, GS; green, WGA;
152 EGGINK ET AL.
vehicle control (Figs. 3B,C). Cells treated with interferon
(IFN)-g as a positive control actively engulfed the micro-
spheres (Fig. 3D). Shown in Figs. 3E–G are macrophage-like
cells in cultures treated with the HPSLK peptide, which ex-
hibited spreading and ruffled edges typical of activated
NPSHPLSG (Figs. 3H and I). A quantitative analysis of the
number of beads per cell, which shows a highly significant
effect of the peptides, is provided in Table 1. In other experi-
ments, streptavidin-coated microspheres, opsonized directly
with antistreptavidin antibodies, were engulfed to the same
extent as shown in Fig. 3, which demonstrated that the effect
of peptides was not specific to the antigen or antibody. In-
ternalization of beads was nearly completely blocked with
50mM cytochalasin D, an inhibitor of phagocytosis,63as as-
sayed by flow cytometry. In these experiments, the inhibi-
tion by cytochalasin D of peptide-stimulated uptake of beads
was similar to that with cells treated with IFN-g (data not
Cytokine response to peptide treatment
Because IFN-g, a proinflammatory cytokine, stimulated
phagocytosis of microspheres, we determined whether the
peptides induced release of cytokines. Cultures of PBMCs
were treated 4h with 100 nM HPSLK or NPSHPLSG peptides
and media were recovered for analysis of cytokines. With
HPSLK, modest increases of 2- to 4-fold, as compared with
untreated controls, were found with I-309, IL-17, TNF-b, and
TIMP2. A nearly 10-fold increase was found in IL-16. The
amounts of these cytokines in the media of cultures treated
with NPSHPLSG were not significantly different from the
untreated control values. No changes were detected in the
release of proinflammatory cytokines such as IL-1a, IL-1b,
IL-2, IL-6, IL-8, TNF-a, or IFN-g with either peptide (data not
Inhibition of HIV-1 replication
IL-16 suppresses HIV-1 replication through inhibition of
viral entry, transcription, and desensitization of HIV-1 re-
ceptors.64–66Thus we determined whether the peptides in-
hibit replication of HIV in cultures of PBMCs. Peptide and the
virus were added at the beginning of a 3-day incubation pe-
riod, with peptide at concentrations from 60 pM to 1mM. At
the end of the 3-day incubation, the cells were washed to
remove unbound virus, peptide, and antiserum, and again
suspended in medium. After an additional 24-h incubation to
allow viral replication, each sample was assayed for protein
p24 to quantify the amount of virus after cells were lysed
by addition of detergent. This assay therefore detected cell-
harbored as well as released virus particles.
HIV-1 SF162, an R5, macrophage-tropic strain of clade B
that is a major subtype in North America, and HIV-1
97ZA009, an R5 strain of clade C that is a major subtype in
Africa, were used as test virus samples. With both subtypes,
the HPSLK peptide alone achieved a reduction in replication
100 nM (Fig. 4A, open symbols). Considerable variability in
the extent of inhibition was observed, yet 20–40% inhibition
was found at concentrations as low as 1 nM. To compare the
cultures to achieve virion-antibody complexes. These North
American patients were presumed to be infected with clade B
HIV-1. When the peptide was added along with antiserum
at a dilution (1:360), which alone caused only about 30%
neutralization, clade B virus expression was reduced to an
undetectable level (Fig. 4A, filled squares). The peptide=
antiserum combination was nearly as effective against the
heterologous clade C strain of the virus (Fig.4A, filled circles).
The effect of the NPSHPLSG peptide on HIV-1 replication
was also assayed. As shown in Fig. 4B, results similar to those
with HPSLK were obtained, although the inhibition with
peptide alone was less. The lower inhibition with the longer
peptide alone possibly resulted from the reduced release of
cytokines by PBMCs as compared withHPSLK. Nevertheless,
in the presence of antiserum the peptide completely inhibited
replication of clade B virus and nearly as strongly inhibited
replication of the clade C virus. Experiments were also per-
formed with peptide concentrations at 1 pM and lower, i.e.,
lower than those shown in Fig. 4. At these concentrations, the
data were not reliable. Inhibition with peptide alone was not
significant and in the presence of antiserum approached that
of the antiserum alone (data not shown).
From the data shown in Fig. 3, we reasoned that the pres-
ence of phagocytic cells in the PBMC cultures was a factor
in the complete inhibition of HIV replication. Therefore,
we determined whether the peptides enhanced inhibition of
as compared with the quadravalent peptide to lectins. Bind-
ing of the quadravalent peptide was set as 100%. The assay
wells contained 25 pmol of the quadravalent peptide, 50 pmol
of the bivalent peptide, and 100 pmol of the monovalent
peptide to provide an equal number of sequences per well.
Lectin designations are?, WGA; &, HP; &, GS; ^, DB.
Binding of bivalent or monovalent HPSLK peptides
INHIBITION OF HIV-1 BY PEPTIDE SUGAR MIMETICS153
HIV replication by antiserum in MT2cells, a T cell line that
lacks phagocytic activity. Addition of undiluted antiserum
completely neutralized the virus. At a dilution of 1:71, the
antiserum alone inhibited viral replication by 35%. Addition
of peptide HPSLK to a final concentration of 10 nM or 1mM
did not increase inhibition (Fig. 5). Similar results were ob-
tained with peptide NPSHPLSG. For comparison, the same
experiment was performed with PBMCs. A greater dilution
(1:360) of the antiserum was required to reduce neutralization
to 30%. However, addition of peptide HPSLK caused com-
plete inhibition of viral replication (Fig. 5). We attribute the
lack of effect of a peptide in with MT2cells to the absence of
antibody-mediated phagocytic activity.
by impairing the cells, their effect on cell viability was deter-
mined. The experimental conditions were similar to those for
1mM or less, the number of viable cells was no less with
PBS or antiserum diluted in PBS (1:360) (Fig. 6). The HPSLK
peptide reduced viability to about 50% at a concentration of
1mM, which is 106-fold greater than a concentration that was
effective in inhibition of HIV replication. Viability was slightly
increased at all concentrations of the NPSHPLSG peptide.
20h, with microspheres for 1h, and then fixed with 2% formalin as described under Materials and Methods. Free micro-
spheres were removed by washing three times with PBS and the samples were examined by microscopy. (A) Control sample
to which vehicle was added. (B, C) Cells treated with 50 nM of an inactive quadravalent peptide containing the sequence
VSNQH. (D) Control sample treated with 100ng=ml IFN-g. (E–G) Cells treated with HPSLK peptide. (H, I) Cells treated with
NPSHPLSG peptide. Phagocytosis experiments were performed four times, with different antibodies, with similar results.
Bar¼10mm for all panels.
Phagocytosis of opsonized microspheres by peptide-treated PBMCs. Cells were incubated with 50 nM peptides for
154EGGINK ET AL.
As demonstrated in Fig. 2, quadravalent peptides bound
lectins approximately two times more strongly than divalent
peptides and 10 times more strongly than monovalent pep-
tides. These differences in binding affinity, with equal num-
bers of sequences, were predicted from entropic factors54,55
and ligand density.56A multivalent peptideis alsoessential to
facilitate cross-linking ofreceptors, which is often requiredfor
transduction of a signal from the cell surface.36When syn-
thesized as a quadravalent molecule, with four branches ex-
tended from a trilysine core, the HPSLK peptide bound with
high affinity to several lectins with differing monosaccharide
specificities. Under the conditions of these assays, peptide
HPSLK had characteristics of a general sugar mimetic. Com-
petition assays indicated that the Kdvalue for binding to
WGA was about 7 nM for the quadravalent peptide. Because
only lectin that was retained after extensive washing in the
greater. The Kdvalues can be considered as only approxi-
mations of binding affinities to cellular receptors. Further
of thelectins wasthe strong inhibition ofbinding to thelectins
by mucin, which contains a variety of complex glycans that
bind more strongly than single sugars.
Peptides with a longer sequence, such as NPSHPLSG, ex-
hibited more restrictive binding. This peptide did not bind
strongly to lectins that bind monosaccharides but bound with
high avidity to those that bind complex glycans. NPSHPLSG
bound more strongly to lectin MAA, which is specific for the
trisaccharide Neu5Ac(a2-3)Gal(b1-4)GlcNAc62than to SNA1,
whereas HPSLK bound more strongly to SNA1, which is
specific for the disaccharide Neu5Ac(a2-6)Gal.60,61SNA1 and
MAA do not bind the monosaccharide, Neu5Ac. We found
that half-maximal binding of the peptides to these lectins was
found in precipitation reactions that these lectins were
inhibited 50% by 12mM Neu5Ac(a2-6)lactose or 500mM
Neu5Ac(a2-3)lactose, respectively. Thus the affinities of the
peptides to these lectins are roughly 103-fold greater than
binding of oligosaccharides and 104-fold greater than that of
monosaccharides.67The affinity with which the NPSHPLSG
peptide binds SNA1 and MAA, considering the extensive
washing steps in the assay, was similar to binding of these
lectins to solid-phase glycan arrays.61
These features suggest that the longer peptides favorably
mimiccomplexglycanssuch asligandsfor avarietyofsiglecs,
which also do not bind significantly to a single Neu5NAc.19,20
Most siglecs are on cells of the immune system, contain an
ITIM in their cytoplasmic domain and are considered to be
to lectins, Carlin et al.68showed that an amino acid sequence
within the N-terminal domain of b protein of group B Strep-
tococcus binds to siglec-5, which contains an ITIM in its cyto-
plasmic domain, and inhibits phagocytosis of the bacterial
cells. In contrast, siglec-14 and siglec-15 have short cytoplas-
mic tails that lack ITIMs and serve as activating receptors in
concert with adaptor proteins such as DAP12, which contains
an ITAM in its cytoplasmic domain.24,25Monocytes, macro-
phages, and dendritic cells also have many C-type lectins on
their surface that are activating receptors.15The possibility
exists that the peptides interact with one or more of these
receptors to stimulate phagocytic activity.
Interestingly, NPSHPLSG appears to be less cytotoxic than
HPSLK, which may result from its more restrictive binding
characteristics. We did not find a significant increase in cy-
tokine release in PBMC cultures in response to treatment with
the longer peptide. However, both peptides were as active in
stimulating phagocytosis of microspheres coated with anti-
HIV antibody as IFN-g (Fig. 3), a highly inflammatory cyto-
kine.69The NPSHPLSG peptide in particular may stimulate
phagocytosis without inducing release of proinflammatory
cytokines. Whereas human anti-HIV antibodies were used in
the HIV replication assays, microspheres were opsonized
with rabbit anti-gp41 or antistreptavidin antibodies, which
indicated that the stimulation of Fc-mediated phagocytosis
was not antigen or antibody specific.
Of particular interest was the complete inhibition of repli-
cation of the clade B strain of HIV-1 in PBMCs in the presence
of pooled antisera obtained from HIV-1 clade B-infected pa-
tients, which alone neutralized only 30% at the dilution used
in these assays. The antiserum appears to contain non-
neutralizing antibodies that nevertheless are capable of virion
clearance when phagocytes are stimulated by the peptides.
Insufficient virus remained at the end of the 3-day treatment
with peptide to provide a detectable amount of protein p24
from clade B virus.
The peptides inhibited replication of HIV-1 over a wide
concentration range in these cultures, with inhibition nearly
as strong at 1 nM as at 1mM, which was indicated by the
shallow slopes of the regression curves for peptide alone in
Fig. 4. Possible explanations for this effect are, first, that given
the Kdof binding to lectins, receptors may have been satu-
ratedat the higherconcentrations of thepeptide. Second,each
quadravalent molecule carries a high local concentration of
the peptide, and thus its effects are expected to be less de-
pendent on stochastic interactions of multiple molecules. As
clustering of receptors is required for many cellular ef-
fects,19,36each multivalent molecule alone can apparently
serve this purpose as the result of ligand density.56
Acute HIV infection elicits both neutralizing antibodies
that block fusion of the virus with host cells and nonneutra-
lizing antibodies,70as reflected in the titration of pooled
antisera shown in Fig. 5. Antibodies bind the virus and
provide the opportunity for phagocytic cells to destroy
Table 1. Phagocytosis of Microspheres Opsonized
with Rabbit Antiserum Raised Against HIV-1 gp41
aPBMC cultures were treated 20h with vehicle (PBS), 50nM
peptide, or 100ng=ml IFN-g and then challenged 1h with micro-
spheres. Cells were fixed with 2% formalin and washed as described
in the legend to Fig. 3. Beads in each cell were counted on
microscope images, with 15cells analyzed for each treatment.
Uptake of beads in cells treated with peptide VSNQH was not
significantly different from the vehicle control (paired Student’s
t-test, p ¼ 0.702). Paired Student’s t-test analyses provided p-values
for the other treatments compared with the control peptide VSNQH.
INHIBITION OF HIV-1 BY PEPTIDE SUGAR MIMETICS 155
(note log scale). Open symbols, peptides alone; closed symbols, peptides added with 1:360 dilution of serum from HIV-
positive patients infected with a clade B virus. Antiserum alone provided 30% inhibition. (A) Peptide HPSLK;(B) peptide
NPSHPLSG.&,&: clade B (strain SF162);*,?: clade C (strain 97ZA009). Each data point is the average of quadruplicate
by polynomial curve fitting: solid line, clade B; dotted line, clade C.
Inhibition of HIV-1 replication in PBMCs cultured with quadravalent peptides at concentrations of 60 pM to 1mM
samples. Results of two experiments are shown for each peptide and viral strain. The lines for peptide alone were generated
156EGGINK ET AL.
virion-antibody complexes (‘‘opsonized’’ virus) by Fc-Fcg re-
ceptor (FcgR)-mediated phagocytosis. A number of research
groups found that viral replication was attenuated by inter-
action of such complexes with FcgR on macrophages and
immature dendritic cells4–9and that activation of macro-
phages in vitro reduced viral load in PBMC cultures.8Hessell
et al.9showed that interaction of the Fc portion of antibodies
with receptors (FcgR) on phagocytes is important in reducing
viral yield from infected cells and, moreover, that the Fc-FcgR
interaction is more important than the complement system.
Nevertheless, concern has been expressed that binding of
complement to antibody-complexed virions may counteract
the immune system and cause complement-dependent, anti-
body-dependent enhancement of infection.71Based on the
lack of detectable p24 at the end of the incubation period,
shown in Fig. 4, our data are consistent with the suggestion4,5
that virion-antibody immune complexes, formed even with
nonneutralizing antibodies, enter a degradative pathway
when phagocytic cells are activated by the peptides. Future
studies will examine in detail the mechanism of this effect.
Our results suggest that the peptides mimic complex gly-
can structures and stimulate antibody-mediated phagocyto-
sis. This activity may be a factor in providing complete
inhibition of HIV-1 replication in vitro. Further studies are
needed to confirm phagocytosis as the mechanism of the ex-
traordinary synergism of the peptides and antibodies. Ad-
ministration of very low concentrations of the sugar-mimetic
peptides alone may have therapeutic potential in AIDS pa-
tients through synergy with autologous HIV antibodies.
series of pooled sera from HIV-positive patients. Each curve is the combined results of two separate experiments. Dilutions of
serum that provided about 30% inhibition were chosen to test the effect of added HPSLK peptide. The two open circles near
the curve for MT2cells indicate results with 10 nM (lower symbol) or 1mM (upper symbol) peptide. Inhibition was 100% with
peptide concentrations between 10 nM and 1mM in PBMC cultures (open square) (see Fig. 4).
Inhibition of replication of HIV-1 (?) strain 23135 in MT2cells or (&) strain SF162 in PBMCs containing a dilution
a 1:360 dilution of HIV-positive serum in PBS (squares).
Cytotoxicity of quadravalent HPSLK (closed symbols)
INHIBITION OF HIV-1 BY PEPTIDE SUGAR MIMETICS 157
Note Added in Proof
Further characterization of the peptides described in this
report is presented in Eggink LL and Hoober JK: Peptide
Insight 2010:2. (This is a new electronic journal.)
L.L.E. and J.K.H. designed the peptides and performed
the lectin binding assays and phagocytosis assays. M.S.
and C.V.H. performed the HIV proliferation assays. J.K.H.
drafted the manuscript and all authors contributed to its
editing. We gratefully acknowledge Daniel Brune and John
Lopez, Proteomics and Protein Chemistry Laboratory, Ar-
izona State University, for peptide synthesis.
Author Disclosure Statement
L.L.E. and J.K.H. declare that they are inventors of tech-
nology contained in this report. Intellectual property has been
assigned to Susavion Biosciences, Inc. in which the inventors
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Address correspondence to:
J. Kenneth Hoober
Susavion Biosciences, Inc.
1615 W. University Drive, Suite 132
Tempe, Arizona 85281
160 EGGINK ET AL.