Increased immunogenicity of human immunodeficiency virus gp120 engineered to express Galalpha1-3Galbeta1-4GlcNAc-R epitopes.
ABSTRACT The glycan shield comprised of multiple carbohydrate chains on the human immunodeficiency virus (HIV) envelope glycoprotein gp120 helps the virus to evade neutralizing antibodies. The present study describes a novel method for increasing immunogenicity of gp120 vaccine by enzymatic replacement of sialic acid on these carbohydrate chains with Galalpha1-3Galbeta1-4GlcNAc-R (alpha-gal) epitopes. These epitopes are ligands for the natural anti-Gal antibody constituting approximately 1% of immunoglobulin G in humans. We hypothesize that vaccination with gp120 expressing alpha-gal epitopes (gp120(alphagal)) results in in vivo formation of immune complexes with anti-Gal, which targets vaccines for effective uptake by antigen-presenting cells (APC), due to interaction between the Fc portion of the antibody and Fcgamma receptors on APC. This in turn results in effective transport of the vaccine to lymph nodes and effective processing and presentation of gp120 immunogenic peptides by APC for eliciting a strong anti-gp120 immune response. This hypothesis was tested in alpha-1,3-galactosyltransferase knockout mice, which produce anti-Gal. Mice immunized with gp120(alphagal) produced anti-gp120 antibodies in titers that were >100-fold higher than those measured in mice immunized with comparable amounts of gp120 and effectively neutralized HIV. T-cell response, measured by ELISPOT, was much higher in mice immunized with gp120(alphagal) than in mice immunized with gp120. It is suggested that gp120(alphagal) can serve as a platform for anti-Gal-mediated targeting of additional vaccinating HIV proteins fused to gp120(alphagal), thereby creating effective prophylactic vaccines.
- SourceAvailable from: Patrice Guillon[Show abstract] [Hide abstract]
ABSTRACT: Background: Over the past century dengue (DENV2), chikungunya (CHIKV) and human parainfluenza (hPiV3) viruses have profoundly impacted on human morbidity, mortality and the economy worldwide. Current therapy options to treat infections of these viruses have severe limitations leading to a continued search for novel drug candidates. Acrostichum aureum L. (Pteridaceae) is a mangrove fern, that has been used as a traditional medicine in Bangladesh and other various countries for a variety of diseases including infection. Objectives: Isolation and structural elucidation of novel antiviral secondary metabolites from the methanol extract of the aerial parts of A. aureum. Materials and methods: The novel phthalate acid ester was isolated (HPLC) and structurally elucidated using 1D and 2D NMR, MS and other spectroscopic methods. The compound was tested for antiviral activity against DENV2 andhPiV3in Vero cells using the fluorescent focus (FFA) assay and against CHIKV virus in LLC-MK2 cells using the plaque-forming unit assay (PFU). The activity of the isolated compound was further compared with its known derivative. Results: In this study, we report on the isolation of a novel phthalic acid ester, 2’’-(methoxycarbonyl)-5’’- methylpentyl 2’-methylhexyl phthalate from the aerial parts of the Bangladeshi mangrove fern Acrostichumaureum and its in vitro antiviral activity. The novel phthalate showed antiviral activity against dengue virus, human parainfluenza virus and chikungunya. The most potent activity was recorded against hPiV3 (EC50 29.4 μM) and was slightly higher than the activity determined for the positive control BCX 2798 (EC50 44 μM). Cellulose acetate phthalate was also evaluated for antiviral activity against these viruses for the first time and was found to be inactive. Both compounds were found to be non-toxic against Vero and LLC-MK2 cells. Conclusion: This study shows that some selected phthalates have potent antiviral activity and should be further investigated as potential novel antiviral agents.Journal of Antivirals and Antiretrovirals 10/2013; 5(6):139-144.
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ABSTRACT: The glycan chains attached to cell surfaces or to single proteins are highly dynamic structures with various functions. The glycan chains of mammals and of some microorganisms often terminate in sialic acids or α-1,3-galactose. Although these two sugars are completely distinct, there are several similarities in their biological and medical importance. First, one type of sialic acid, N-glycolylneuraminic acid, and the galactose bound by an α-1,3-linkage to LacNAc, that forms an α-gal epitope, were both eliminated in human evolution, resulting in the production of antibodies to these sugars. Both of these evolutionary events have consequences connected with the consumption of foods of mammalian origin, causing medical complications of varying severity. In terms of ageing, sialic acids prevent the clearance of glycoproteins and circulating blood cells, whereas cryptic α-gal epitopes on senescent red blood cells contribute to their removal from circulation. The efficiency of therapeutic proteins can be increased by sialylation. Another common feature is the connection with microorganisms since sialic acids and α-gal epitopes serve as receptors on host cells and can also be expressed on the surfaces of some microorganisms. Whereas, the sialylation of IgG antibodies may help to treat inflammation, the expression of the α-gal epitope on microbial antigens increases the immunogenicity of the corresponding vaccines. Finally, sialic acids and the α-gal epitope have applications in cancer immunotherapy. N-glycolylneuraminic acid is a powerful target for cancer immunotherapy, and the α-gal epitope increases the efficiency of cancer vaccines. The final section of this article contains a brief overview of the methods for oligosaccharide chain synthesis and the characteristics of sialyltransferases and α-1,3-galactosyltransferase.Central European Journal of Biology 10/2012; 7(5). · 0.63 Impact Factor
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ABSTRACT: Production of long-lived, high affinity humoral immunity is an essential characteristic of successful vaccination and requires cognate interactions between T and B cells in germinal centers. Within germinal centers, specialized T follicular helper cells assist B cells and regulate the antibody response by mediating the differentiation of B cells into memory or plasma cells after exposure to T cell-dependent antigens. It is now appreciated that local immune responses are also essential for protection against infectious diseases that gain entry to the host by the mucosal route; therefore, targeting the mucosal compartments is the optimum strategy to induce protective immunity. However, because the gastrointestinal mucosae are exposed to large amounts of environmental and dietary antigens on a daily basis, immune regulatory mechanisms exist to favor tolerance and discourage autoimmunity at these sites. Thus, mucosal vaccination strategies must ensure that the immunogen is efficiently taken up by the antigen presenting cells, and that the vaccine is capable of activating humoral and cellular immunity, while avoiding the induction of tolerance. Despite significant progress in mucosal vaccination, this potent platform for immunotherapy and disease prevention must be further explored and refined. Here we discuss recent progress in the understanding of the role of different phenotypes of B cells in the development of an efficacious mucosal vaccine against infectious disease.Current pharmaceutical biotechnology 12/2013; · 3.40 Impact Factor
JOURNAL OF VIROLOGY, July 2006, p. 6943–6951
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 80, No. 14
Increased Immunogenicity of Human Immunodeficiency Virus gp120
Engineered To Express Gal?1-3Gal?1-4GlcNAc-R Epitopes
Ussama Abdel-Motal, Shixia Wang, Shan Lu, Kim Wigglesworth, and Uri Galili*
Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605
Received 13 February 2006/Accepted 1 May 2006
The glycan shield comprised of multiple carbohydrate chains on the human immunodeficiency virus (HIV)
envelope glycoprotein gp120 helps the virus to evade neutralizing antibodies. The present study describes a
novel method for increasing immunogenicity of gp120 vaccine by enzymatic replacement of sialic acid on these
carbohydrate chains with Gal?1-3Gal?1-4GlcNAc-R (?-gal) epitopes. These epitopes are ligands for the
natural anti-Gal antibody constituting ?1% of immunoglobulin G in humans. We hypothesize that vaccination
with gp120 expressing ?-gal epitopes (gp120?gal) results in in vivo formation of immune complexes with
anti-Gal, which targets vaccines for effective uptake by antigen-presenting cells (APC), due to interaction
between the Fc portion of the antibody and Fc? receptors on APC. This in turn results in effective transport
of the vaccine to lymph nodes and effective processing and presentation of gp120 immunogenic peptides by APC
for eliciting a strong anti-gp120 immune response. This hypothesis was tested in ?-1,3-galactosyltransferase
knockout mice, which produce anti-Gal. Mice immunized with gp120?galproduced anti-gp120 antibodies in
titers that were >100-fold higher than those measured in mice immunized with comparable amounts of gp120
and effectively neutralized HIV. T-cell response, measured by ELISPOT, was much higher in mice immunized
with gp120?galthan in mice immunized with gp120. It is suggested that gp120?galcan serve as a platform for
anti-Gal-mediated targeting of additional vaccinating HIV proteins fused to gp120?gal, thereby creating
effective prophylactic vaccines.
Many of the studies of recombinant protein and DNA hu-
man immunodeficiency virus (HIV) vaccines in primate mod-
els or in clinical trials report that these vaccines have not been
found as yet to be satisfactory in eliciting a sterilizing protec-
tive immune response against infection with HIV or simian
immunodeficiency virus (SIV) (3, 22, 31, 40). An effective
prophylactic HIV vaccine has to induce a strong memory (an-
amnestic) immune response for the rapid production of neu-
tralizing antibodies and a rapid cytotoxic T-lymphocyte (CTL)
response. Such a combined immune response will enable pre-
vention of cell infection primarily by neutralizing anti-gp120
(anti-Env) antibodies and destruction of infected host cells in
early stages following transmission of the virus, when the num-
ber of infected cells is relatively low. In the absence of a rapid
immune response, the infecting virus multiplies and mutates
before anti-Env antibodies are produced in titers high enough
to prevent spreading of the infectious virus into a large number
of cells. These mutations in envelope glycoproteins enable
HIV to escape the neutralizing antibodies without losing re-
ceptor binding activity (3–5, 22, 27, 31, 40, 41, 53, 58).
A major component on the envelope of HIV, which contrib-
utes to the masking of the virus from the immune system and
which hinders the effective uptake of gp120 vaccines, is the
multiple carbohydrate chains on this envelope glycoprotein
(19, 30, 34). The HIV gp120 is quite unique among viral gly-
coproteins as it has a very high number of N (asparagine
[Asn])-linked carbohydrate chains which form a “glycan
shield” for this virus (58). There are ?24 N-linked carbohy-
drate chains on this glycoprotein with the size of 479 amino
acids (30). As many as 13 to 16 of these carbohydrate chains
are of the complex type which are capped with sialic acid (SA)
(left chain in Fig. 1), and the rest are of the high-mannose type
(19, 30, 34). The size of each of these carbohydrate chains is
approximately 30% (?60 Å) of the diameter of the protein
portion of the gp120 molecule in its globular form. Since they
are hydrophilic, these carbohydrate chains protrude from the
gp120 molecule and seem to contribute to the protection of
HIV against neutralizing antibodies. This protective role of the
multiple carbohydrate chains can be inferred from isolate
clones of HIV type 1 (HIV-1) in AIDS patients, where at least
half of the mutations in gp120 (i.e., the env gene) result in the
appearance of new N-glycosylation sites (i.e., Asn-X-Ser/Thr)
(58). These de novo-expressed carbohydrate chains provide a
“glycan shield” that protects the virus from neutralizing anti-
We have developed a method to convert the carbohydrate
chains on gp120 into a means for effectively targeting vacci-
nating gp120 to antigen-presenting cells (APC), thereby in-
creasing their immunogenicity. This is achieved by enzymatic
engineering of the complex-type carbohydrate chains on gp120
for the replacement of SA with ?-gal (Gal?1-3Gal?1-4GlcNAc-R)
epitopes, as illustrated in Fig. 1. The in situ targeting of vac-
cinating gp120 molecules expressing ?-gal epitopes (referred
to as gp120?gal) to APC is mediated by the natural anti-Gal
antibody. This natural antibody constitutes ?1% of serum
immunoglobulin G (IgG) (20 to 100 ?g/ml serum) (16), and it
interacts specifically with ?-gal epitopes on glycolipids and
glycoproteins (10, 15). The ?-gal epitope is absent in humans
but is abundantly synthesized by the glycosylation enzyme
?-1,3-galactosyltransferase (?1,3GT) within the Golgi appara-
* Corresponding author. Mailing address: Department of Medicine,
University of Massachusetts Medical School, 364 Plantation Street,
LRB, Worcester, MA 01605. Phone: (508) 856-4188. Fax: (508) 856-
4106. E-mail: Uri.Galili@umassmed.edu.
tus of cells in nonprimate mammals and in New World mon-
keys (10, 12, 18). Humans, apes, and Old World monkeys lack
an active ?1,3GT gene but produce the anti-Gal antibody in
large amounts (10, 12, 18).
Anti-Gal interacts very effectively with ?-gal epitopes in
vivo. This can be inferred from xenotransplantation studies. In
vivo binding of anti-Gal to ?-gal epitopes on transplanted pig
heart or kidney is the main cause for the rapid rejection of such
grafts in humans and in Old World monkeys (9, 11, 20, 45).
This rejection is mediated by complement that is activated as a
result of anti-Gal binding to ?-gal epitopes on the graft endo-
thelial cells. However, even if complement activation is inhib-
ited, xenografts are rejected because of binding of various
effector cells with Fc? receptors (Fc?R) (e.g., monocytes/mac-
rophages, neutrophils, and NK cells) to the Fc portion of
anti-Gal on xenograft cells and destruction of these cells by the
antibody-dependent cell cytotoxicity mechanism (11, 57). In
addition, because of its effective interaction with Fc?R, anti-
Gal can opsonize tumor cells expressing ?-gal epitopes for very
effective uptake by various APC including macrophages and
dendritic cells (DC) which express these receptors, thereby
increasing the immunogenicity of tumor vaccines (13, 28, 33).
We hypothesize that immunization with gp120?gal(i.e.,
gp120 engineered to carry ?-gal epitopes) will elicit a much
stronger anti-gp120 immune response than immunization with
gp120. The natural anti-Gal antibody, diffusing into the vacci-
nation site from ruptured capillaries, forms immune complexes
with gp120?galand effectively targets the vaccinating molecules
to Fc?R of APC, thereby greatly increasing uptake and trans-
port of gp120 by APC to the lymph nodes. Moreover, the
binding of gp120?gal/anti-Gal immune complexes to Fc?R on
dendritic cells further stimulates differentiation and matura-
tion of these APC into professional APC that present gp120
peptides on both class I and class II major histocompatibility
complex molecules for effective activation of gp120-specific
CD8?and CD4?T cells, respectively (8, 32, 42, 44, 46).
Engineering gp120 into gp120?galis achieved by an enzy-
matic reaction combining the activities of neuraminidase and
recombinant ?1,3GT, as described in Fig. 1. The only available
nonprimate mammalian experimental model in which immu-
nogenicity of gp120?galcould be analyzed is the knockout
mouse for the ?1,3GT gene (KO mouse), in which the ?1,3GT
gene was disrupted by targeted insertion of the neomycin re-
sistance gene (51, 52). This mouse mimics the relevant human
immune characteristics, as it lacks ?-gal epitopes and it can
produce anti-Gal in titers comparable to those in humans (29,
36, 38, 39, 50, 52). The studies described below indicate that
the anti-gp120 immune response in KO mice immunized with
gp120?galcan be ?100-fold higher than that in mice immu-
nized with comparable amounts of gp120.
MATERIALS AND METHODS
Supplies. The gp120BALprotein produced in CHO cells was received as a
generous gift from the NIH AIDS Research and Reference Reagent Program.
Neuraminidase extracted from Vibrio cholerae was purchased from Sigma (St.
Louis, MO). Recombinant ?1,3GT was produced in the authors’ laboratory in
the Pichia pastoris expression system according to a procedure that was previ-
ously described (7). The monoclonal anti-Gal antibody designated M86 was
obtained in tissue culture supernatants of hybridoma M86 cells, as previously
described (14). Horseradish peroxidase (HRP)-conjugated anti-mouse IgG and
anti-mouse IgM antibodies were purchased from Accurate Laboratories (West-
bury, NY). HRP-conjugated Bandeiraea (Griffonia) simplicifolia IB4 (BS lectin,
specific for ?-gal epitopes) was purchased from Vector Laboratories (Burlin-
game, CA). Synthetic ?-gal epitopes linked to bovine serum albumin (?-gal BSA)
were purchased from Dextra Laboratories (Reading, United Kingdom). Human
anti-Gal was isolated from healthy human serum by using an affinity column of
synthetic ?-gal epitopes linked to silica beads (Synsorb, Alberta, Canada), as
previously described (12). Mouse anti-Gal was also isolated on an affinity column
by the same method as previously described (29), using serum from KO mice that
were repeatedly immunized with pig kidney membrane (PKM) homogenate, in
order to elicit anti-Gal production in the mice. The Ribi adjuvant, trehalose
dicorynomycolate, was purchased from Corixa (Hamilton, MT).
Mice and immunization procedures. Mice used were ?1,3GT KO mice on an
H-2bxdgenetic background (52) which are bred and maintained at the animal
facility of UMass Medical School. Studies were performed with both males and
females and found to yield similar results. All experiments with mice were
performed according to AAALAC guidelines. The mice were immunized intra-
peritoneally three times with 50 mg PKM homogenate for inducing anti-Gal
production in titers similar to those of anti-Gal in humans (titers of 1:200 to
1:2,000 as measured by enzyme-linked immunosorbent assay [ELISA] with ?-gal
BSA as solid-phase antigen) (38, 39, 48–50). Following the demonstration of
anti-Gal IgG production, the mice received a subcutaneous injection of gp120 or
gp120?galin Ribi adjuvant. The injection was repeated after 2 weeks. The anti-
gp120 immune response was evaluated 17 days after the second injection.
Enzymatic engineering of gp120 to express ?-gal epitopes. The enzymatic
reactions described in Fig. 1 are performed simultaneously in an enzyme buffer
containing 0.1 M MES (methyl ethyl morpholinosulfonate), pH 6.0, and 25 mM
MnCl2as previously described for the synthesis of ?-gal epitopes on influenza
virus hemagglutinin (24), on the bovine serum glycoprotein fetuin (7), and on the
human serum glycoprotein ?1, acid glycoprotein (49). The terminal SA is re-
moved by neuraminidase (1 mU/ml), and ?-gal epitopes are synthesized on
gp120 by recombinant ?1,3GT (30 ?g/ml) and UDP-Gal (1 mM). The two
enzymes are mixed in the same solution buffer and incubated with gp120 (1
mg/ml) for 2 h at 37°C. At the end of incubation, the gp120?galmolecules are
purified from the mixture of the enzymatic reaction by an affinity Sepharose
column of Bandeiraea simplicifolia IB4 (BS lectin), which specifically binds ?-gal
epitopes (59). No other substances in the enzyme reaction bind to this lectin. The
gp120?galbound to the BS-Sepharose beads is eluted with 100 mM of ?-methyl
galactoside which competes with ?-gal epitopes for binding to the lectin. This
enzymatic reaction and the subsequent affinity column process result in the
isolation of ?97% of the processed gp120 as gp120?galmolecules.
ELISAs. Anti-Gal titers in mice immunized with PKM and the production of
anti-gp120 antibodies were determined by ELISA as previously described (24, 29,
35, 36, 39). Briefly, ELISA wells were coated with ?-gal BSA or gp120 molecules
(10 ?g/ml) overnight at 4°C. Plates were washed once with phosphate-buffered
saline (PBS) and blocked with 1% BSA in PBS. Serum samples at various
dilutions were plated at 50-?l aliquots in the wells for 2 h at 24°C. After washing,
HRP-coupled goat anti-mouse IgG was added for 1 h. The color reactions were
developed with orthophenylene diamine, and absorbance was measured at 492
nm. In assays using the monoclonal anti-Gal antibody M86 (14), HRP–anti-
mouse IgM was used as secondary antibody.
Analysis of neutralizing anti-gp120 antibodies. The assay for neutralizing
antibodies has been previously described (56) and is based on the study by
Montefiori et al. (37). The assay was performed with the HIV-1 lab strain MN.
The neutralization of HIV-1 lab strain MN was measured by the killing assay of
the human T-cell lymphoma line MT-2. The virus stock of HIV-1 MN is pro-
duced in H9 cells. Virus in 50-?l aliquots containing 1,000 50% tissue culture
infective doses was added to multiple dilutions of test sera and incubated at 37°C
for 1 h in microtiter tissue culture wells. The T-cell lymphoma MT-2 cells (5 ?
104cells in 100 ?l) were added to each well. Infection of the cells by HIV leads
to extensive syncytium formation and virus-induced cell killing in 5 to 7 days in
the absence of neutralizing antibodies. Neutralization was measured by staining
viable cells with Finter’s neutral red and measuring their adhesion to poly-L-
lysine-coated wells in ELISA plates. Percent protection was determined by cal-
culating the difference in absorbance at 540 nm between test wells (containing
cells, serum sample, and virus) and virus control wells (cells and virus) and dividing
this result by the difference in absorbance between cell control wells (cells only) and
virus control wells. Neutralization was measured at a time when virus-induced cell
killing in virus control wells was greater than 70% but less than 100%. Neutralizing
antibody titers are expressed as the reciprocals of the serum dilutions required to
protect 50% of cells from virus-induced killing. The background neutralization data
measured with control sera from nonimmunized mice were subtracted from the data
in the mice immunized with either gp120 or gp120?gal.
6944 ABDEL-MOTAL ET AL.J. VIROL.
APC for ELISPOT assay. Bone marrow-derived DC were prepared as de-
scribed previously (1). Briefly, bone marrow cells were cultured in RPMI me-
dium containing granulocyte-macrophage colony-stimulating factor and interleu-
kin-4. On day 5, immature dendritic cells were pulsed with 100 ?M of gp120
protein for 18 h to allow protein processing. Cells were then washed and used for
IFN-? ELISPOT assay. ELISPOT assays for gamma interferon (IFN-?)-se-
creting cells were performed with a commercial kit (Mabtech, Ohio), according
to the manufacturer’s protocol. Briefly, 96-well ELISPOT plates were coated
with 100 ?l/well of anti-IFN-? monoclonal antibody AN18 overnight at 4°C. The
plates were washed with PBS and blocked with PBS containing 10% fetal calf
serum for 30 min at room temperature. Freshly isolated splenocytes (2 ? 105
cells per well) were plated in triplicate together with dendritic cells (2 ? 104)
prepulsed with gp120 protein as described above. After overnight incubation at
37°C in 5% CO2, cells were removed by washing with PBS and aliquots of 100 ?l
of anti-IFN-?–biotin (monoclonal antibody R4-6A2; Mabtech) were added to
each well for 2 h at room temperature. The plates were then washed with PBS,
and 100 ?l of streptavidin-alkaline phosphatase was added per well and incu-
bated for 1 h at room temperature. After washing with PBS, 100 ?l of chromo-
genic substrate (NBT-plus; Mabtech) was added to each well for 15 min to allow
color development and formation of spots. The color reaction was stopped by the
addition of water. Wells were then air dried, and spots were counted with an
ELISPOT automated reader system (performed by Zellnet, Fort Lee, NJ). Cal-
culated frequencies were based on the average of the triplicate wells. The results
are expressed as gp120-specific IFN-?-secreting T cells per 106splenocytes, i.e.,
the number of spots after subtraction of the spot number in corresponding
control wells that lack pulsed dendritic cells.
Synthesis of ?-gal epitopes on gp120 by recombinant ?1,3GT.
Replacement of SA on the gp120 carbohydrate chains with
?-gal epitopes is achieved by a two-step enzymatic reaction
within one solution (Fig. 1). The studied recombinant gp120
was of the HIVBALstrain and was produced in CHO cells that
were transformed with the corresponding codon-optimized env
gene. The SA was removed from the carbohydrate chains by
neuraminidase to expose the penultimate N-acetyllactosamine
residues (Gal?1-4GlcNAc-R) on the multiple complex type
carbohydrate chains of gp120. The N-acetyllactosamines ex-
posed on the carbohydrate chains function as an acceptor for
recombinant ?1,3GT, which links to them terminal ?1-3-galac-
tosyl residues, to form ?-gal epitopes. The sugar donor pro-
viding galactose to ?1,3GT is UDP-galactose (UDP-Gal). The
latter enzymatic reaction is identical to that occurring within
the Golgi apparatus of nonprimate mammalian cells for the
synthesis of ?-gal epitopes. The recombinant ?1,3GT used for
the generation of gp120?galis produced in the expression sys-
tem of the yeast Pichia pastoris transformed by a New World
monkey ?1,3GT gene (7), which was originally cloned from
marmoset cells (25). Because of the de novo-expressed multi-
ple ?-gal epitopes, gp120?galcan be purified from the mixture
of the enzymatic reaction by an affinity Sepharose column of
Bandeiraea simplicifolia IB4 (BS lectin), which interacts specif-
ically with these epitopes (59). This enzymatic reaction and the
subsequent column separation process result in the purifica-
tion of ?97% of the gp120 as gp120?galmolecules. As ex-
pected, the enzymatic manipulation did not result in changes in
the size of gp120 as shown by sodium dodecyl sulfate-poly-
acrylamide gel electrophoresis (SDS-PAGE) (Fig. 2, upper
FIG. 1. Synthesis of ?-gal epitopes on gp120. SA residues capping the N-linked carbohydrate chains of the complex type on gp120 (left chain)
are removed by neuraminidase (middle chain). ?-gal epitopes are synthesized by linking of galactosyls (Gal) from the sugar donor UDP-galactose
(UDP-GAL), due to the catalytic activity of recombinant ?1,3GT. These ?-gal epitopes on immunizing gp120?galreadily bind in situ the natural
anti-Gal IgG molecules, thus forming immune complexes that target the vaccinating gp120?galto APC.
VOL. 80, 2006 INCREASED IMMUNOGENICITY OF gp120 WITH ?-gal EPITOPES 6945
panel). The de novo expression of ?-gal epitopes on gp120?gal
could be demonstrated in Western blots that are immuno-
stained with human anti-Gal, mouse anti-Gal, and the ?-gal
epitope-specific BS lectin (Fig. 2, lower panel). The two anti-
bodies and the lectin all interact specifically with ?-gal
epitopes, readily bound to gp120?galbut not to the original
The approximate number of the de novo-synthesized ?-gal
epitopes on the gp120?galmolecules could be estimated by
measuring the binding of the monoclonal anti-Gal antibody
M86 (15) to different amounts of this molecule dried in ELISA
wells and by comparison with this antibody binding to a stan-
dard glycoprotein expressing 10 synthetic ?-gal epitopes, ?-gal
BSA. The two glycoproteins were dried at various concentra-
tions as solid-phase antigens in ELISA wells, and binding of
the monoclonal anti-Gal was measured in the different wells.
As shown in Fig. 3, gp120?galbound comparable amounts of
monoclonal anti-Gal in wells containing fourfold-less protein
than in the wells containing ?-gal BSA. Since ?-gal BSA size is
?70% of that of gp120?gal, the data imply that there are ?3-
fold-more ?-gal epitopes on gp120?galthan on ?-gal BSA, i.e.,
?30 ?-gal epitopes/molecule. As indicated above, gp120 has 13
to 16 N-linked carbohydrate chains of the complex type, each
with two branches (antennae) (Fig. 1) (19, 30, 34). Therefore,
these data imply that most SA residues on gp120 were replaced
by ?-gal epitopes as a result of the enzymatic reactions with
neuraminidase and ?1,3GT. As expected, the monoclonal anti-
Gal did not bind to the original gp120, since this glycoprotein
lacks ?-gal epitopes (Fig. 3).
Similarities between anti-Gal produced in humans and in
?1,3GT-knockout mice (KO mice). Analysis of anti-Gal-medi-
ated targeting of gp120?galversus gp120 vaccines requires an
appropriate experimental animal model that produces anti-
Gal. The ?1,3GT-knockout mouse (KO mouse) is the only
available nonprimate animal model because this mouse, in
contrast to all other nonprimate mammals, lacks ?-gal epitopes.
KO mice naturally produce anti-Gal in titers much lower than
those in humans. However, anti-Gal production in these mice
can be increased to titers comparable to those in humans when
they are immunized with xenogeneic cell membranes express-
ing ?-gal epitopes, such as PKMs (36, 38, 39, 50). The similarity
in anti-Gal activities in KO mice and in humans could be
demonstrated in an ELISA measuring this antibody’s binding
to ?-gal BSA. As shown in Fig. 4A there is a similarity in
binding curves of anti-Gal at various dilutions of human or
mouse serum samples. Furthermore, KO mice produce anti-
Gal that has characteristics similar to those of human anti-Gal.
Anti-Gal in humans is found as IgM, IgG, and IgA classes (16,
23), and anti-Gal IgG is found in all subclasses, IgG1 being the
predominant one (43). Similarly, KO mice used in this study
produced anti-Gal IgG that comprised all subclasses (IgG1,
IgG2a, IgG2b, and IgG3), with IgG1 being the predominant
subclass (Fig. 4B). IgG4 is not shown, since mice do not pro-
duce this subclass. Whereas anti-Gal IgM is also produced in
mice, as in humans (23, 49), no significant production of anti-
Gal IgA was detected in the PKM-immunized KO mice (Fig.
4B). This class of anti-Gal is readily found in the serum and in
body secretions in humans (23). Because of the overall simi-
larities in the characteristics of anti-Gal IgG and anti-Gal IgM
in humans and in KO mice, these mice can serve as an appro-
priate model for determining whether formation of immune
complexes between gp120?galand anti-Gal has any effect on
the elicited anti-gp120 immune response.
Anti-gp120 antibody response in KO mice immunized with
gp120?galor gp120. KO mice with confirmed production of
anti-Gal IgG in titers comparable to those in humans were
immunized subcutaneously twice at a 2-week interval with 5 ?g
of either gp120 or gp120?gal. Synthetic trehalose dicorynomy-
colate (TDM-Ribi) was used as adjuvant. This adjuvant was
chosen because it has been approved by the FDA for experi-
mental use in humans. Anti-gp120 antibody production was
tested by ELISA in serum samples obtained 17 days after the
second immunization. The titer is defined as the serum dilution
FIG. 2. Synthesis of ?-gal epitopes on gp120 by recombinant
?1,3GT as demonstrated by SDS-PAGE and Western blotting. Upper
gel: Coomassie staining of SDS-PAGE gel. Lower gels: Western blot
stained with human anti-Gal, mouse anti-Gal, and the ?-gal epitope-
specific BS lectin. Note that the antibodies and the lectin bind only to
FIG. 3. Evaluation of ?-gal epitope expression on gp120?gal. The
figure shows binding of the monoclonal anti-Gal M86 to gp120,
gp120?gal, and ?-gal BSA that expresses 10 synthetic ?-gal epitopes, as
measured by ELISA with different amounts of glycoproteins coating
the ELISA wells.
6946ABDEL-MOTAL ET AL.J. VIROL.
yielding 50% maximal binding (i.e., ?1.5 optical density [OD]
units). All mice immunized with gp120?galdisplayed extensive
production of anti-gp120 antibodies with titers ranging be-
tween 1:320 and 1:2,560. Anti-gp120 antibody production was
much lower in mice immunized with the original (i.e., unpro-
cessed) gp120. Three of these mice displayed marginal produc-
tion of anti-gp120 antibodies at the lowest serum dilution of
1:20 (0.5 to 1.0 OD), whereas the remaining two immunized
mice displayed no significant anti-gp120 antibody production
(?0.5 OD) (Fig. 5A). These findings imply that gp120?galis at
least 100-fold more immunogenic than gp120 in its ability to
induce the production of anti-gp120 antibodies. The differen-
tial immunogenicity of gp120?galversus gp120 was less distinct
with lower or higher vaccine doses (Fig. 5B and 5C). Two
immunizations with 0.5 ?g gp120?galelicited a strong anti-
gp120 antibody response in two of the five mice tested and less
response in the remaining three mice (Fig. 5B). Nevertheless,
all mice in this group displayed higher anti-gp120 antibody
responses than mice vaccinated with 0.5 ?g gp120, in which no
significant antibody response was detected (Fig. 5B). Immu-
nization with 50 ?g gp120 (10-fold higher than in Fig. 5A)
resulted in distinct production of anti-gp120; however, immu-
nizations with equal amounts of gp120?galresulted in a ?30-
fold-higher antibody response on average (Fig. 5C). It is not
clear at present whether the overall higher anti-gp120 antibody
response in mice immunized with 5 ?g versus 50 ?g of
gp120?galis the result of the use of different gp120?galbatches
or is because of an inherent higher immunogenicity of the 5-?g
dose. Evaluation of the kinetics of anti-gp120 antibody pro-
duction indicated that this activity could be detected in the
serum of the immunized mice only 2 weeks after the second
immunization, whereas no such antibodies were identified af-
ter the first immunization with 120?galor gp120, even at the
high dose of 50 ?g (not shown).
The increased immune response to gp120?galis associated
with the presence of anti-Gal antibody molecules which form
immune complexes with the immunizing glycoprotein. This can
be inferred from analysis of anti-gp120 antibody response in
wild-type C57BL/6 mice, i.e., the parental strain of KO mice
which cannot produce anti-Gal, despite repeated PKM immu-
nizations, because they express ?-gal epitope (49). Wild-type
mice immunized twice with either 5 ?g gp120 or 5 ?g gp120?gal
displayed low titers of anti-gp120 antibodies, as there are no
antibodies in these mice that can target the vaccine to APC
Neutralizing activity of anti-gp120 antibodies in immunized
mice. The major potential protective activity of anti-gp120
antibodies is in neutralizing HIV, thereby preventing infection
of the host cells. Thus, it was of interest to determine whether
anti-gp120 antibodies produced in KO mice immunized with
gp120?galcan display neutralizing activity. The neutralizing
activity of these antibodies was evaluated with the HIV-1 lab
strain MN which is convenient for manipulation in the labo-
ratory and for the analysis of neutralizing activity by antibod-
ies. The neutralization was measured by the killing assay of the
human T-cell lymphoma line MT-2 (37, 56). The analysis of the
neutralizing activity with HIVMNis a conservative approach
for evaluating the production of protective antibodies against
HIVBAL. Although the gp120BALsequence may differ by a few
amino acids from that of gp120MN, demonstration of neutral-
izing antibodies with HIVMNwill imply an even higher neu-
tralizing activity against HIVBAL. In accord with the low titers
of anti-gp120 antibodies in the serum from the mice immu-
nized with gp120 (Fig. 5A), serum from these mice displayed
no neutralizing activity above that of the nonimmunized mice
(mice 1 to 6 in Fig. 6). In contrast, the serum from the mice
immunized with gp120?gal(mice 7 to 12 in Fig. 6) displayed a
very effective neutralization activity, which was similar to that
observed in the positive control of rabbit serum containing
anti-HIV neutralizing antibodies (originating from a rabbit
receiving multiple immunizations with gp120) (56). These data
demonstrate a correlation between the high titer of anti-gp120
antibody production as a result of immunization with gp120?gal
and the neutralizing activity of the elicited antibodies in the
Analysis of gp120-specific T cells by ELISPOT. Detection of
IFN-?-secreting cells in the immunized mice was determined
by ELISPOT assay as described in Materials and Methods and
reference 2. The number of IFN-?-secreting cells in gp120?gal-
immunized mice was significantly higher than that in gp120-
FIG. 4. Characteristics of mouse anti-Gal in comparison to human anti-Gal. A. Comparison of anti-Gal IgG activity in human sera (F) and sera
of mice immunized with PKMs (E). Data are for 3 out of 30 humans and 30 KO mice with similar results. B. Classes and subclasses of anti-Gal
in KO mice as measured by ELISA with ?-gal BSA as solid-phase antigen. The antibody activity was measured at a serum dilution of 1:100. Data
from 4 representative mice out of 15 with similar results are shown.
VOL. 80, 2006 INCREASED IMMUNOGENICITY OF gp120 WITH ?-gal EPITOPES6947
immunized mice that were tested simultaneously. The ELISPOT
wells with lymphocytes from three out of six mice in each group
are shown in Fig. 7A. The numerical values of IFN-?-secreting
T cells per 1 ? 106cells in six mice tested in each group, after
subtraction of the number of spots in the corresponding con-
trol wells lacking the gp120 pulsed dendritic cells, are pre-
sented in Fig. 7B. Despite the variability between the individ-
ual mice, the number of spots representing gp120-specific T
cells is much higher in the group immunized with gp120?gal
than in the group of mice immunized with gp120. The mean
number of spots in gp120?gal-immunized mice was calculated
to be 332 spots/106cells, whereas the mean number of spots in
mice immunized with gp120 was only 23 spots/106cells. These
findings imply that T-cell activation against the gp120 peptides
was much higher in gp120?gal-immunized mice than in gp120-
This study demonstrates a method for increasing the immu-
nogenicity of gp120 by replacing its multiple SA residues with
?-gal epitopes. These epitopes can bind the natural anti-Gal
antibody (present in all humans as 1% of IgG), when injected
FIG. 5. Elicited anti-gp120 antibodies in response to immunization with gp120 or gp120?gal. The figure shows production of anti-gp120
antibodies in KO mice immunized twice at 2-week intervals (A to C) or in wild-type (WT) mice (D), immunized with either gp120 (E) or gp120?gal
(F). (A) 5.0 ?g/vaccine; (B) 0.5 ?g/vaccine; (C) 50 ?g/vaccine; (D) 5.0 ?g/vaccine. Note that KO mice in panels A to C immunized with gp120
produced anti-gp120 antibodies in low titers or completely lacked such antibodies, whereas a significant increase in anti-gp120 antibody production
was observed in mice immunized with gp120?gal. In contrast, no differences in anti-gp120 antibody production are detected in panel D in wild-type
mice immunized with the two glycoproteins, as the wild-type mice are incapable of producing anti-Gal despite repeated PKM immunizations.
FIG. 6. HIV neutralization activity in mice immunized with gp120?gal
or with gp120. The figure shows the titer of neutralization activity in
various mice immunized twice with 5 ?g gp120 (mice 1 to 6) or
gp120?gal(mice 7 to 12). Titer is defined as the reciprocal of the serum
dilution displaying 50% neutralization.
6948 ABDEL-MOTAL ET AL.J. VIROL.
as a vaccine in humans. The formation of immune complexes
with anti-Gal results in targeting of the vaccinating gp120?gal
molecules to APC, thereby inducing an effective anti-gp120
immune response. The incubation of gp120 with an enzyme
mixture of neuraminidase and recombinant ?1,3GT and with
the sugar donor UDP-Gal results in synthesis of multiple ?-gal
epitopes on most N-linked carbohydrate chains of the complex
type on gp120, as indicated by the subsequent extensive bind-
ing of the monoclonal anti-Gal antibody (Fig. 3). The in-
creased immunogenicity of gp120?galwas demonstrated in the
only nonprimate mammalian experimental model available for
studies of anti-Gal-mediated immune response, the ?1,3GT-
knockout mouse (KO mouse). Our previous studies demon-
strated in this model increased immunogenicity of tumor vac-
cines consisting of tumor cells engineered to express ?-gal
epitopes (28). In tumor vaccine studies, the efficacy of vaccines
expressing ?-gal epitopes can be demonstrated by the immune
protection following challenge of the mouse with live tumor
cells. The present study demonstrates increased immunogenic-
ity of a soluble protein expressing ?-gal epitopes, the HIV
envelope glycoprotein gp120. The increased production of
anti-gp120 antibodies in response to vaccination with gp120?gal
versus vaccination with gp120 was observed in all three doses
of 0.5, 5, and 50 ?g per vaccine. The greatest difference, of
?100-fold, in the antibody response was observed in mice
immunized with 5 ?g of the glycoprotein. Similarly, we ob-
served a parallel increase in T-cell response, as assessed by
ELISPOT. However, we could not analyze immune protection
from challenge postvaccination as there are no HIV strains
that are infective in mice.
The principle of increasing immunogenicity of a given anti-
gen by 10- to 1,000-fold, by complexing the antigen with its
corresponding antibody, was demonstrated with a variety of
antigens, including tetanus toxoid (21, 32), hepatitis B virus
antigen (6), and Eastern equine encephalomyelitis virus anti-
gen (26). Accordingly, recent studies demonstrated that im-
mune complexes between SIV and anti-SIV antibodies were
targeted to APC, resulting in enhanced cross-presentation of
SIV peptides, as indicated by effective activation of cytotoxic T
cells by MHC class I-presented peptides, in SIV-infected mon-
keys (55). As expected, enzymatic destruction of the Fc portion
of the anti-SIV antibodies, or blocking of the Fc?R on APC,
abrogated this enhancing effect of immune complexes (55).
Similarly, natural antibodies in mice were found to function as
an endogenous adjuvant forming immune complexes with
Leishmania vaccine and inducing a strong CD8?-T-cell re-
sponse against the intracellular form of the Leishmania para-
site (47). Thus, the interaction between the Fc portion of the
opsonizing antibody and Fc?R on APC is considered to be the
most effective mechanism by which APC identify and internal-
ize antigens that should be targeted for an effective immune
response (54). The same principle applies to vaccines that
express ?-gal epitopes and thus form immune complexes with
anti-Gal antibodies which can target the vaccine to APC in any
immunized individual. This anti-Gal-mediated targeting to
APC is supported by previous in vitro studies of inactivated
influenza virions that express ?-gal epitopes and form immune
complexes with anti-Gal. These virions displayed a 10-fold-
higher uptake by APC and subsequent processing and presen-
tation of envelope hemagglutinin peptides than virions lacking
FIG. 7. ELISPOT analysis for IFN-? secretion in mice immunized with gp120 or gp120?gal. A. Actual wells with splenocytes from three mice,
each tested in triplicate (vertical lanes) in the absence or presence of gp120-pulsed DC. B. Presentation of ELISPOT data for six mice immunized
twice with 5 ?g gp120 (mice 1 to 6) and six mice immunized twice with 5 ?g gp120?gal(mice 7 to 12), as the number of spots per 106splenocytes.
VOL. 80, 2006INCREASED IMMUNOGENICITY OF gp120 WITH ?-gal EPITOPES6949
?-gal epitopes and incubated with anti-Gal (17). It should be
stressed that anti-Gal is the only antibody in humans that can
serve for this purpose of targeting vaccines to APC. This is
because anti-Gal is the only natural antibody known to be
produced ubiquitously in humans as ?1% of IgG (16). Thus,
any particulate or soluble vaccine that expresses ?-gal epitopes
will form immune complexes with anti-Gal and will be targeted
for effective uptake by APC (13).
In view of the ability of gp120 to mutate during infection and
evade the detrimental effect of neutralizing antibodies, vacci-
nation only with gp120 may not suffice for conferring resistance
to HIV infections in large populations (3–5, 22, 27, 31, 40, 41,
53, 58). Other viral proteins such as tat, rev, p17, and p24 may
also be used as vaccines eliciting a cellular immune response
for destruction of HIV-infected cells. However, because of
poor targeting to APC, immunogenicity of these proteins also
may be low. The effective anti-Gal-mediated targeting of
gp120?galto APC may be further exploited for effective target-
ing of other HIV proteins to APC, in order to induce a pro-
tective cellular immune response. This can be achieved by
fusion of tat, rev, p17, or p24 to gp120 and enzymatic conver-
sion of the SA residues on the carbohydrate chains of gp120
into ?-gal epitopes as in Fig. 1. Thus, vaccination with
gp120?galthat is fused to each of these proteins is likely to
produce high titers of anti-gp120 antibodies, as well as high
CTL activity against cells infected by HIV.
As indicated above, KO mice are the only nonprimate mam-
mal that produces anti-Gal and thus can serve as a model for
anti-Gal-mediated targeting of vaccines to APC. Anti-Gal pro-
duction in these mice is achieved by immunization with PKMs.
This mouse-produced anti-Gal is an elicited antibody, whereas
anti-Gal in humans is a natural antibody. Nevertheless, they
share similar characteristics in their class and subclass distri-
bution (Fig. 4) and in biological activities. Both human and
mouse anti-Gal mediate hyperacute xenograft rejection and
induce antibody-dependent cell cytotoxicity and phagocytosis
of the various antigens (9, 11, 20, 45). Despite these similarities,
demonstration of primate anti-Gal ability to target gp120?galto
APC will require studies in a monkey model. Since Old World
monkeys (e.g., rhesus monkeys, cynomolgus monkeys, and ba-
boons) naturally produce anti-Gal in titers comparable to
those of humans (12), future studies of immunogenicity in
monkeys immunized with SIV gp120?galand with gp120?gal
fused to other viral proteins will enable evaluation of the effi-
cacy of these vaccines in eliciting a protective immune re-
sponse against challenge with SIV.
This study was supported by NIH grant AI58749.
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