Synthesis and evaluation of monophosphoryl lipid A derivatives as fully synthetic self-adjuvanting glycoconjugate cancer vaccine carriers

Article abstract of DOI:10.1039/c4ob00390j

A fully synthetic carbohydrate-based cancer vaccine is an attractive concept, but an important topic in the area is to develop proper vaccine carriers that can improve the immunogenicity and other immunological properties of tumor-associated carbohydrate antigens (TACAs). In this context, four monophosphoryl derivatives of Neisseria meningitidis lipid A were synthesized via a highly convergent and effective strategy and evaluated as vaccine carriers and adjuvants. The conjugates of these monophosphoryl lipid A (MPLA) derivatives with a modified form of the sTn antigen were found to elicit high titers of antigen-specific IgG antibodies, indicating a T cell-dependent immune response, in the absence of an external adjuvant. It was concluded that MPLAs could be utilized as potent vaccine carriers and built-in adjuvants to create fully synthetic self-adjuvanting carbohydrate-based cancer vaccines. The lipid composition and structure of MPLA were shown to have a significant influence on its immunological activity, and among the MPLAs examined, natural N. meningitidis MPLA exhibited the most promising properties. Moreover, Titermax Gold, a conventional vaccine adjuvant, was shown to inhibit, rather than promote, the immunological activity of MPLA conjugates, maybe via interacting with MPLA.

Full text of DOI:10.1039/c4ob00390j

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Biomolecular Chemistry
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Synthesis and evaluation of monophosphoryl lipid
A derivatives as fully synthetic self-adjuvanting
glycoconjugate cancer vaccine carriers†
Cite this: Org. Biomol. Chem., 2014,
12, 3238
Zhifang Zhou,‡ Mohabul Mondal,‡ Guochao Liao‡ and Zhongwu Guo*
A fully synthetic carbohydrate-based cancer vaccine is an attractive concept, but an important topic in
the area is to develop proper vaccine carriers that can improve the immunogenicity and other immuno-
logical properties of tumor-associated carbohydrate antigens (TACAs). In this context, four monophos-
phoryl derivatives of Neisseria meningitidis lipid A were synthesized via a highly convergent and effective
strategy and evaluated as vaccine carriers and adjuvants. The conjugates of these monophosphoryl lipid A
(MPLA) derivatives with a modified form of the sTn antigen were found to elicit high titers of antigen-
specific IgG antibodies, indicating a T cell-dependent immune response, in the absence of an external
adjuvant. It was concluded that MPLAs could be utilized as potent vaccine carriers and built-in adjuvants
to create fully synthetic self-adjuvanting carbohydrate-based cancer vaccines. The lipid composition and
structure of MPLA were shown to have a significant influence on its immunological activity, and among
the MPLAs examined, natural N. meningitidis MPLA exhibited the most promising properties. Moreover,
Titermax Gold, a conventional vaccine adjuvant, was shown to inhibit, rather than promote, the immuno-
logical activity of MPLA conjugates, maybe via interacting with MPLA.
Received 20th February 2014,
Accepted 11th March 2014
DOI: 10.1039/c4ob00390j
www.rsc.org/obc
on the Gram negative bacterial cell surface.8 Lipid A is of great
importance in that it not only serves as an anchor to attach
Introduction
Fully synthetic glycoconjugate cancer vaccines are currently a bacterial O-polysaccharides to the bacterial cell membrane8
hot topic, since they have well-defined structures, reproducible but also is a potent toxin that can stimulate strong immune
physical, chemical and biological properties, and promising responses in eukaryotic hosts.9 It binds specifically to the toll
immunological activities.1–7 To develop functional conjugate or interleukin-1 receptor domain-containing adaptor-inducing
cancer vaccines, a vital issue is the carrier molecule. An ideal interferon-β (TRIF) of toll-like receptors (TLRs) in association
vaccine carrier should be rather small to be synthetically with MD88 to induce a downstream signaling cascade, stimu-
manageable and highly immunoactive to be able to improve late the release of cytokines and chemokines, such as tumor
the immunogenicity and promote T cell-dependent immunity necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 and inter-
of tumor-associated carbohydrate antigens (TACAs) that are feron-β (IFN-β), and upgrade immune cell expression.10 Even
often poorly immunogenic and T cell-independent. While though the immunostimulatory activity of lipid A can be extre-
several vaccine carriers have been explored for this purpose,1–7 mely useful, its pro-inflammatory and septic properties are a
this paper presents a new type of vaccine carrier derived from serious problem.11
lipid A for fully synthetic self-adjuvanting carbohydrate-based
cancer vaccines.
Regardless of the source, all lipid As contain the highly con-
served construct 1 (Fig. 1) with several lipids, varying consider-
Lipid A is the conserved hydrophobic core of lipopoly- ably in chain length, saturation, number and distribution,
saccharides (LPSs) – the main component and virulent factor linked to the N-2-, N-2′-, O-3- and O-3′-positions and two phos-
phate groups linked to the O-1- and O-4′-positions of the β-1,6-
linked disaccharide of D-glucosamine (GlcNH2). Biological
assays demonstrated that the number, structure and position
of lipids and the degree of phosphorylation are important
factors determining the bioactivity of lipid A.12–14 A typical
lipid A that exhibits a full spectrum of immunological and
endotoxic activities consists of six lipids that can be distribu-
ted symmetrically (3 + 3), such as in the lipid A of Neisseria
Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit,
MI 48202, USA. E-mail: zwguo@chem.wayne.edu; Fax: +1-313-577-5060;
Tel: +1-313-577-2557
†Electronic supplementary information (ESI) available: Experimental, 1H, 13C,
and 2D NMR spectra and MS spectra of all new compounds. See DOI:
10.1039/c4ob00390j
‡These authors contributed equally to the present paper.
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Fig. 1 The conserved construct of lipid A (1) and the structures of N. meningitidis lipid A (2) and designed MPLA derivatives and their sTn conjugates
(3–6).
meningitidis (2, Fig. 1),15 or asymmetrically (4 + 2), such as in
the lipid A of Escherichia coli, on the two GlcNH2 units. The
two phosphate groups are essential for the endotoxic activity
of lipid A. However, modification of the O-1-position, for
example, removal of the phosphate group at this position to
generate monophosphoryl lipid A (MPLA), was shown to
greatly reduce its endotoxicity with little impact on its
immunostimulatory activity.16 Thus, MPLA has emerged as a
potential adjuvant used in human vaccine formulation.17,18
Scheme 1 The retrosynthesis for 3–6.
However, the optimal lipid A structure required for binding to
the cell surface receptors is different from the structure
required for the activation of the host immune system, sup-
ported by the finding that most biosynthetic precursors of
Results and discussion
In our synthetic design for target molecules 3–6 (Scheme 1),
the key intermediates were fully protected MPLA derivatives
7–10, which had a 2-azidoethyl group at the reducing end of
the disaccharide backbone. Global deprotection of 7–10 would
afford free MPLA derivatives that can be explored as indepen-
dent vaccine adjuvants. On the other hand, the azido group in
7–10 could be selectively reduced to form a primary amine that
would facilitate conjugation with sTnNPhAc or other TACAs to
generate conjugate vaccines. In return, the MPLA derivatives
would be constructed from a common disaccharide inter-
mediate 11 having the 2,2′-N- and 3,3′-O-positions differently
protected from other positions to enable regioselective de-
protection and acylation later on. Moreover, the 4′,6′-O-posi-
tions in 11 were protected with a benzylidene group that can
be regioselectively opened to expose the 4′-O-position for the
introduction of the conserved phosphoryl group.
lipid A are nontoxic and that the toxicity of lipid A has mul-
tiple structural requirements.19 Consequently, a systematic
study on the structure–activity relationship of MPLA is necess-
ary to discover proper vaccine carriers and adjuvants.
In this regard, many lipid A and MPLA derivatives have
been prepared and evaluated in the literature.13,14,17,20–31 In
association with our efforts to develop fully synthetic carbo-
hydrate-based cancer vaccines, we synthesized a monophos-
phoryl analog32 of N. meningitidis lipid A and coupled it to
N-phenylacetyl GM3, a modified form of TACA.33 The resulting
conjugate alone induced promising immune responses in
animals in the absence of any external adjuvant, suggesting
that, as an immunostimulant, MPLA was functional not only
as a vaccine carrier but also as a built-in adjuvant.34 Inspired
by this discovery, here we have designed and synthesized three
other monophosphoryl analogs of the lipid A of N. meningiti-
dis, containing lipids of different chain lengths and linkages
as well as different patterns of lipidation (Fig. 1). To investigate
the immunological properties of these MPLA derivatives as
vaccine carriers and adjuvants, we conjugated them with
N-phenylacetyl sTn (sTnNPhAc), a modified TACA that is cur-
rently studied for anticancer vaccine development,35 and exam-
ined in mice the specific immune responses induced by the
resulting MPLA-sTnNPhAc conjugates 3–6 (Fig. 1). Through
these studies, we anticipated to identify novel vaccine carriers
and adjuvants with improved immunological properties useful
for the development of fully synthetic glycoconjugate cancer
vaccines.
The synthesis started with the preparation of properly
protected D-glucosamine derivatives 12 and 13 (Scheme 2),
employed as a glycosyl donor and acceptor, respectively,
according to reported procedures.32 Glycosylation of 12 with
13 was realized with N-iodosuccinimide (NIS) and silver triflate
(AgOTf) as promoters, and was unexpectedly slow and took
two days to complete. The doublet signal of H-1′ at δ 5.88 in
1
the H NMR spectrum of the resulting disaccharide 11 gave a
coupling constant (J1′,2′) of 8.8 Hz, which confirmed the β-con-
formation of the newly generated glycosidic linkage. Treatment
of 11 with hydrazine hydrate in refluxing ethanol removed
both N-phthalyl and O-acetyl groups simultaneously to form
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Scheme 2 Synthesis of the key intermediates 7–10.
14, which was ready for the introduction of lipids. In the of the phosphate group to the 4′-O-position. All of the inter-
meantime, the chiral lipids 15 and 18–20 (Scheme 2) required mediates involved in the syntheses were fully characterized by
for these syntheses were also prepared according to reported 1H, 13C and correlation NMR spectroscopy and by MALDI-TOF
procedures32 with commercial (R)-epichlorohydrine as the MS.
stating material.
To be readied for conjugation with sTnNPhAc, MPLA
For lipid installation (Scheme 2), the free amino groups in derivatives 7–10 were treated with Zn and acetic acid to selec-
14 were acylated first by reacting with 15 under the influence tively reduce the azido group (Scheme 3). The resulting amines
of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI). 30–33 were then acylated with succinic acid anhydride
Despite the presence of two free hydroxyl groups in 14, the to produce 34–37, which had a free carboxylic acid group
reaction was fast and selective for the amino groups, probably attached to the disaccharide reducing end. For coupling with
owing to their higher reactivity, to produce 16 in an excellent sTnNPhAc derivative 42 that carried a free amino group, acids
yield (94%). The two NH signals as doublets at δ 6.19 (J 34–37 were converted to active esters 38–41 by reacting with
4.8 Hz) and 5.99 (J 6.8 Hz) as well as the down-field shift of p-nitrophenol and EDCI. These reactions were fast, and the
1
the H-2 and H-2′ signals in the H NMR spectrum of 16 con- active esters formed were stable enough to enable silica gel
firmed the desired N-acylation. Compound 16 was employed column purification. Subsequently, 38–41 were coupled with
as a common intermediate for the synthesis of all MPLA 42 in DMF. These reactions were smooth and clean to afford
derivatives designed. Acylation of the 3-O- and 3′-O-positions 43–46 that were readily purified by silica gel column chromato-
in 16 with lauric acid (17) and EDCI required DMAP as a cata- graphy and by preparative TLC. Finally, catalytic hydrogeno-
lyst to afford 21. It was observed that the reaction to introduce lysis of the benzyl ether linkages in 43–46 under a H2
the last lipid chain into either the 3-O- or the 3′-O-position was atmosphere in the presence of 10% Pd/C afforded the desired
very slow and could be complete only after prolonged reaction glycoconjugates 3–6, which were confirmed by both NMR and
time, probably due to the further increased steric hindrance MS. Thereafter, 3–6 were subjected to biological evaluation in
after introducing the third lipid into one of these two posi- mice.
tions. Similarly, the coupling reactions between 16 and fatty
Immunological investigation of the MPLA-sTnNPhAc conju-
acids 18–20 in the presence of EDCI and DMAP generated gates 3–6 was carried out with female C57BL/6 mouse, a well-
22–24, respectively. Regioselective opening of the benzylidene characterized inbred strain for vaccine evaluation and
ring in 21–24 was accomplished using NaBH3CN under acidic comparable to the human in immune reactions.33 To improve
conditions to result in 25–28, which had a free hydroxyl group the solubility of these conjugates in buffer, they were incorpor-
at the 4-O′-position. The regiochemistry of these intermediates ated in liposomes formed by using 1,2-distearoyl-sn-glycero-3-
was confirmed by the observation of a down-field shift of 4′-H phosphocholine and cholesterol. Delivering glycoconjugate
in the 1H NMR spectra of the acetylation products as well as by a vaccines in liposomal forms can further improve their immu-
similar down-field shift of the 4′-H observed in the O-phosphoryl nogenicity.36,37 For immunization, the liposomal preparations
products of the next step. Phosphorylation of 25–28 was achieved of 3–6 were each injected subcutaneously (s.c.) to a group of
by the phosphoramidite approach in a two-step one-pot manner six mice. In the meantime, groups of mice were simul-
to afford the key intermediates 7–10. As mentioned above, sig- taneously inoculated with emulsions of 3–6 liposomes and
nificant down-field shift of the H-4′ signals was observed with Titermax Gold to evaluate the potential influence of an exter-
1
the H NMR spectra of 7–10 (δ 4.3–4.5), as compared to that of nal adjuvant on the activities of these glycoconjugate vaccines.
25–28 (δ 3.5–3.6), which further substantiated the attachment The adopted vaccination schedule was to inject into each
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Scheme 3 Assembly of the target conjugates 3–6.
mouse 0.1 mL of a vaccine preparation containing ca. 3 μg of
sTnNPhAc on days 1, 14, 21 and 28, respectively. Each mouse
was subjected to bleeding on day 0 before the first injection
(used as blank controls) and on days 27 and 38 after immuniz-
ation. The blood samples were treated according to standard
protocols to prepare antisera for the analysis of sTnNPhAc-
specific antibodies by enzyme-linked immunosorbent assay
(ELISA) with the human serum albumin (HSA) conjugate of
sTnNPhAc (sTnNPhAc-HSA)38 as a capture antigen. In addition
to total antibodies, antibody isotypes such as IgG1, IgG2a,
IgG3 and IgM were also individually assessed. Antibody titers
were calculated from linear regression analysis of the curves of
the optical density (OD) value against serum dilution number,
and were defined as the dilution number yielding an OD value
of 0.2.34
The ELISA results of antisera obtained with 3 without the
use of an external adjuvant (Fig. 2A) revealed that the conju-
gate itself provoked a strong sTnNPhAc-specific immune
response in mice. More importantly, in addition to IgM
antibody, a high titer of IgG3 and some IgG1 antibodies were
also observed, suggesting
a T cell-dependent immune
response.39,40 Interestingly, 3 plus Titermax Gold failed to
stimulate a significant antibody response (Fig. 2B), although
IgM antibodies were observed. These results suggest that the
external adjuvant had an inhibitory effect on the T cell-
mediated immune response against 3 but a relatively small
influence on the B cell response. The results agreed well with
our previous report about other MPLA-TACA conjugates.34
The ELISA results of 4 (Fig. 2C and 2D) were similar to that
of 3 in terms of the type of immune responses provoked and
the influence of Titermax Gold. However, the IgM and IgG
Fig. 2 ELISA results of day 38 antisera of mice immunized with 3 alone
(A), 3 plus Titermax Gold (B), 4 alone (C), 4 plus Titermax Gold (D), 5
alone (E), and 6 alone (F), respectively. The titers of various sTnNPhAc-
specific antibodies are displayed. Each dot represents the antibody titer
of an individual mouse, and the black bar shows the average antibody
titer of a group of six mice.
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Fig. 3 Competitive ELISA results of the pooled mouse antisera obtained
with conjugates 4, 5, 6, and 4 plus adjuvant. The OD values at 405 nm
reflected the levels of total antibodies bound to the sTnNPhAc-HSA-
coated ELISA plates in the presence of specified concentrations of 42.
antibody titers for 4 were much higher than that for 3. More-
over, 4 stimulated a strong immune response in all of the
mice. Another interesting finding was that 4 also induced a
significant level of IgG1 antibody which is usually observed
with neoglycoprotein vaccines. The ELISA results of 5 (Fig. 2E)
and 6 (Fig. 2F) were similar to that of 3 and 4, but their anti-
body titers were lower than that of 4 and higher than that of 3.
In all of these cases, Titermax Gold showed inhibition of the
immune responses against the conjugate vaccines.
To further verify that the detected antibodies were indeed
sTnNPhAc-specific, a competitive ELISA experiment for the
pooled mouse antisera obtained with 4, 5, 6, and 4 plus adju-
vant was performed. In this study, various concentrations
(0, 0.00152, 0.0152, 0.152, 1.52, 15.2 and 152 μM) of free
sTnNPhAc (42) were added to compete with sTnNPhA-HSA
attached to the ELISA plate for antibody binding. Again, alka-
line phosphatase-linked goat anti-mouse kappa antibody was
employed as the secondary antibody. The OD values at 405 nm
reflected the levels of total antibodies bound to the sTnNPhAc-
HSA-coated plates. These studies (Fig. 3) have revealed that 42
had concentration-dependent inhibition of antibody binding
to the plates and that, at concentrations higher than 152 μM,
42 could essentially completely inhibit the binding, proving
that the elicited antibodies were indeed specific to the
sTnNPhAc antigen.
The above ELISA results disclosed that the MPLA conjugates
3–6 alone, that is, in the absence of any external adjuvant,
provoked a robust antigen-specific immune response in mice.
More importantly, they stimulated high titers of IgG3 and IgG1
antibodies, indicating a T cell-mediated immune response
desirable for cancer immunotherapy.39,40 Therefore, all of the
synthetic MPLA derivatives could act both as a vaccine carrier
and as an adjuvant to effectively enhance the immunogenicity
of sTnNPhAc and to efficiently promote a T cell-dependent
immune response toward TACAs.18,34,41 These properties of
MPLA should be particularly useful for the design and devel-
opment of new fully synthetic carbohydrate-based conjugate
vaccines.
Fig. 4 Comparison of the average antibody titers of sTnNPhAc-specific
kappa (total), IgG3, and IgM antibodies in day 38 antisera of mice immu-
nized with 3, 4, 5, and 6.
to improve their immunogenicity. However, we showed in this
work that it had an inhibitory impact on the immunological
activities of 3–6. As Titermax Gold mainly inhibited the elicita-
tion of IgG antibodies, it seemed to mainly affect the T cell-
mediated immune response. We proposed that Titermax Gold
might interact with MPLA in the conjugate vaccine to prevent
MPLA from binding to cell surface TLRs and affect its engage-
ment in T cell-mediated immunological pathways. Another
hypothesis is that Titermax Gold might interact with MPLA to
affect vaccine delivery to the lymph system or antigen present-
ing cells required for effective T cell-mediated immunity.43,44
This discovery and further detailed studies may help under-
stand vaccine adjuvants, such as their binding sites, functional
mechanisms and so on.
Despite the fact that 3–6 stimulated similar patterns of
immune responses in mice, they did show differences in
immunological activity. For example, conjugate 4 elicited
much higher and more consistent titers of both total anti-
bodies and IgG3 antibody than 3, 5 and 6 (Fig. 4). Moreover,
all of the mice showed strong immune responses to 4 but not
to other conjugates. Clearly, 4 was the best vaccine among the
MPLA conjugates investigated.
Structurally, 3–6 were different only in their MPLA moiety,
and their antisera were obtained by means of the same immu-
nization protocol and schedule. Consequently, any difference
in their immunological activity should reflect the impact of
the MPLA structure. Conjugate 4 contained the monophos-
phoryl form of natural lipid A of N. meningitidis (H44/76
strain).15 In 3, the two free hydroxyl groups on the lipid chains
of the MPLA moiety were removed; 5 and 6 were different in
that their MPLA contained different lengths and different
numbers of lipid chains than that of 4. As all these conjugates
had essentially the same immunological profile, it seems that
Titermax Gold is a commercial adjuvant that is commonly
used with vaccines such as carbohydrate-protein conjugates42
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the free hydroxyl groups on the lipid chains and the length Gold, which is a very important topic in cancer vaccine
and number of lipid chains of MPLA had a quantitative, rather immunology.
than qualitative, impact on its biological activities. From what
was said, it is clear that the hydroxyl groups on the lipid
chains play an important role in the MPLA interaction with its
receptors, as 3 was significantly less potent than both 4 and 5.
Elongating the length of lipids at the 3-O- and 3′-O-positions
and additional lipid chains linked to these lipids seem to have
a relatively small impact, although such changes did result in
reduced immunological activity and some inconsistence of
Experimental section
Synthesis of the target glycoconjugates 3–6
Detailed experimental procedures for these syntheses and
analytical data of compounds are described in the ESI†
document.
immune responses in individual mice.
Preparation of liposomes of glycoconjugates 3–6
The protocol was similar to that reported in the literature.34 In
brief, a mixture of a specific conjugate 3–6 (0.474 µmol), 1,2-
distearoyl-sn-glycero-3-phosphocholine (2.44 mg, 3.08 µmol),
Conclusion
and cholesterol (0.92 mg, 2.37 µmol) (in a 10 : 65 : 50 molar
ratio) was dissolved in CH2Cl2 and MeOH (1 : 1, v/v, 2 mL) in a
round-bottom flask. The solvents were then removed under
vacuum to form a thin lipid film on the flask wall, which was
hydrated by adding 2.0 mL of HEPES buffer (20 mM, pH 7.5)
containing NaCl (150 mM) and shaking the mixture under an
argon atmosphere at 40 °C for 1 h. The suspension was finally
sonicated for 1 min to obtain the desired liposomes.
In summary, a convergent and efficient strategy was developed
for the synthesis of MPLA derivatives suitable for conjugation
with carbohydrate antigens, and was employed to synthesize
four monophosphoryl derivatives of N. meningitidis lipid
A. These MPLA derivatives were coupled with sTnNPhAc to
form fully synthetic glycoconjugate cancer vaccines. The strat-
egy should be generally applicable in preparing other MPLA
derivatives and MPLA-carbohydrate conjugates. Studies on the
resulting MPLA-sTnNPhAc conjugates revealed that they eli-
cited strong and T cell-dependent immune responses without
the use of any external adjuvant. Our previous work revealed
that antisera derived from mice immunized with MPLA conju-
gates could effectively bind to and kill cancer cells metaboli-
cally engineered to express the corresponding antigen.34 MPLA
has thus been demonstrated to be a useful platform for the
development of new vaccine carriers and adjuvants and for the
development of novel types of fully synthetic carbohydrate-
based cancer vaccines with self-adjuvanting properties. Our
results have also revealed that MPLA derivatives containing six
lipid chains exhibited more potent immunostimulatory activi-
ties than those with eight lipid chains (conjugate 6) and that
the lipid structure and length had a significant impact on the
immunology of MPLA. The monophosphoryl form of natural
N. meningitidis lipid A was found to have the most promising
immunological properties and its sTnNPhAc conjugate elicited
the most potent and the most consistent T cell-dependent
anti-sTnNPhAc immunity. As a result, the MPLA moiety in 4
was identified as the first generation of optimized vaccine car-
riers and adjuvants that is under further optimization and
additional investigation.
Immunization of mice
Each group of six female C57BL/6 mice was inoculated on day
1 by subcutaneous (s.c.) injection of 0.1 mL of the liposomal
solution of a specific glycoconjugate containing 10 µg of the
carbohydrate antigen or by injection of an emulsion of the
liposomal solution (0.1 mL) of a specific glycoconjugate with
Titermax Gold adjuvant (0.05 mL) prepared according to the
manufacturer’s protocol. Following the initial immunization,
the mice were boosted 3 times on day 14, day 21 and day 28 by
s.c. injection of the same conjugate and by means of the same
immunization protocol. Mouse blood samples were collected
prior to the initial immunization on day 0 and after immuniz-
ation on day 27 and day 38, and were clotted to obtain antisera
that were stored at −80 °C before use. The animal protocol
(#A 03-10-11) for this investigation was approved by the Insti-
tutional Animal Care and Use Committee (IACUC) of Wayne
State University, and all animal experiments were performed
in compliance with the relevant laws and institutional
guidelines.
ELISA
On the other hand, Titermax Gold was found to inhibit the ELISA plates were treated with 100 µL of a solution of the
immunological activity of MPLA-sTnNPhAc conjugates, sTnNPhAc-HSA conjugate (2 µg mL−1) dissolved in coating
whereas it has the opposite influence on the activity of buffer (0.1 M bicarbonate, pH 9.6) at 37 °C for 1 h, which was
protein-sTnNPhAc conjugates.35,38 It is proposed that Titermax followed by treatment with a blocking buffer and washing 3
Gold may interact with MPLA to affect its binding to cell times with phosphate-buffered saline (PBS) containing 0.05%
surface receptors and/or its delivery to the lymph system or Tween-20 (PBST). Then, the pooled or an individual mouse
antigen presenting cells. It is anticipated that these issues may antiserum with serial half-log dilutions from 1 : 300 to
be clarified by studies utilizing labeled MPLA derivatives and 1 : 656 100 in PBS was added to the coated plates (100 µL per
conjugates, the results of which should be useful for under- well) and incubated at 37 °C for 2 h. The plates were washed
standing the immunostimulatory and adjuvant activities with PBS and incubated at rt for another hour with a 1 : 1000
and the functional mechanisms of MPLA and Titermax diluted solution of alkaline phosphatase linked goat anti-
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mouse kappa, IgM or IgG2a antibody or a 1 : 2000 diluted solu-
tion of alkaline phosphatase linked goat anti-mouse IgG1 or
J. R. Baldridge, P. Probst, R. J. Ulevitch, D. H. Persing and
R. M. Hershberg, J. Biol. Chem., 2004, 279, 4440–4449.
IgG3 antibody (100 µL per well), respectively. Finally, the 14 S. Kanegasaki, K. Tanamoto, T. Yasuda, J. Y. Homma,
plates were washed with PBS and developed with 100 µL of a
p-nitrophenylphosphate (PNPP) solution (1.67 mg mL−1 in
buffer) for 30 min at rt for colorimetric readout using a micro-
M. Matsuura, M. Nakatsujka, Y. Kumazawa, A. Yamamoto,
T. Shiba, S. Kusumoto, M. Imoto, H. Yoshimura and
T. Shimamoto, J. Biochem., 1986, 99, 1203–1210.
plate reader at 405 nm wavelength. For titer analysis, OD 15 V. A. Kulshin, U. Zahringer, B. Lindner, C. E. Frasch,
values were plotted against antiserum dilution values, and a
best-fit line was obtained. The equation of this line was used
C. M. Tsai, B. A. Dmitriev and E. T. Rietschel, J. Bacteriol.,
1992, 174, 1793–1800.
to calculate the dilution value at which an OD of 0.2 was 16 E. Ribi, J. Cantrell, T. Feldner, K. Myers and J. Peterson,
achieved, and the antibody titer was calculated at the inverse Microbiol., 1986, 9–13.
of the dilution value. For the competitive ELISA experiments, 17 J. Baldridge, K. Myers, D. Johnson, D. Persing, C. Cluff and
before the addition of antiserum (80 µL) to each well of the
plate, a solution of free sTnNPhAc derivative 42 (20 µL) was
added to reach the final concentration of 0, 0.00152, 0.0152,
R. Hershberg, in Vaccine Adjuvants, ed. C. J. Hackett and
D. A. J. Harn, Humana Press Inc., Totowa, NJ, 2006, pp.
235–255.
0.152, 1.52, 15.2 and 152 μM, respectively. The remaining oper- 18 C. R. Casella and T. C. Mitchell, Cell. Mol. Life Sci., 2008,
ations were the same as those described above.
65, 3231–3240.
19 K. Takayama, N. Qureshi, E. Ribi and J. Cantrell, Rev. Infect.
Dis., 1984, 6, 439–443.
20 Y. Fukase, Y. Fujimoto, Y. Adachi, Y. Suda, S. Kusumoto
and K. Fukase, Bull. Chem. Soc. Jpn., 2008, 81, 796–819.
Acknowledgements
This research work was supported by an NIH/NCI grant (R01 21 Z. Jiang, W. A. Budzynski, D. Qiu, D. Yalamati and
CA95142).
R. R. Koganty, Carbohydr. Res., 2007, 342, 784–796.
22 A. Zamyatina, H. Sekljic, H. Bradeb and P. Kosmaa, Tetrahe-
dron, 2004, 60, 12113–12137.
23 B. Santhanam, M. A. Wolfert, J. N. Moore and G.-J. Boons,
Chem. – Eur. J., 2004, 10, 4798–4807.
Notes and references
1 Q. Wang and Z. Guo, Curr. Opin. Chem. Biol., 2009, 13, 608– 24 Y. Watanabe, K. Miura, M. Shiozaki, S. Kanai, S. Kurakata
617.
and M. Nishijimac, Carbohydr. Res., 2003, 338, 47–54.
25 A. V. Demchenko, M. A. Wolfert, B. Santhanam, J. N. Moore
and G.-J. Boons, J. Am. Chem. Soc., 2003, 125, 6103–6112.
2 F. Peri, Chem. Soc. Rev., 2013, 42, 4543–4556.
3 A. Kaiser, N. Gaidzik, T. Becker, C. Menge, K. Groh, H. Cai,
Y. Li, B. Gerlitzki, E. Schmitt and H. Kunz, Angew. Chem., 26 T. Mochizuki, Y. Iwano, M. Shiozaki, S. Kurakata, S. Kanaic
Int. Ed., 2010, 49, 3688–3692. and M. Nishijima, Tetrahedron, 2000, 56, 7691–7703.
4 Z. Huang, L. Shi, J. Ma, Z. Sun, H. Cai, Y. Chen, Y. Zhao 27 D. A. Johnson, D. S. Keegan, C. G. Sowell, M. T. Livesay,
and Y. Li, J. Am. Chem. Soc., 2012, 134, 8730–8733.
5 V. Lakshminarayanana, P. Thompsonb, M. A. Wolfertb,
T. Buskasb, J. M. Bradleyc, L. B. Pathangeyc, C. S. Madsena,
P. A. Cohenc, S. J. Gendlera and B.-J. Boons, Proc. Natl.
Acad. Sci. U. S. A., 2012, 109, 261–266.
C. J. Johnson, L. M. Taubner, A. Harris, K. R. Myers,
J. D. Thompson, G. L. Gustafson, M. J. Rhodes, J. T. Ulrich,
J. R. Ward, Y. M. Yorgensen, J. L. Cantrell and
V. G. Brookshire, J. Med. Chem., 1999, 42, 4640–4649.
28 K. Fukase, Y. Fukase, M. Oikawa, W. Liu, Y. Suda and
S. Kusumoto, Tetrahedron, 1998, 4033–4050, (and refer-
ences therein).
6 A. M. Abdel-Aal, D. El-Naggar, M. Zaman, M. Batzloff and
I. Toth, J. Med. Chem., 2012, 55, 6968–6974.
7 H. Cai, Z. Sun, Z. Huang, L. Shi, Y. Zhao, H. Kunz and 29 W. J. Christ, P. D. McGuinness, O. Asano, Y. Wang,
Y. Li, Chem.–Eur. J., 2013, 19, 1962–1970.
8 C. Erridge, E. Bennett-Guerrero and I. R. Poxton, Microbes
Infect., 2002, 4, 837–851.
M. A. Mullarkey, M. Perez, L. D. Hawkins, T. A. Blythe,
G. R. Dubuc and A. L. Robidoux, J. Am. Chem. Soc., 1994,
116, 3637–3638.
9 C. A. Alving and M. Rao, Vaccine, 2008, 26, 3036–3045.
10 M. A. Dobrovolskaia and S. N. Vogel, Microbes Infect., 2002,
4, 903–914.
30 A. J. Ulmer, H. Heine, W. Feist, S. Kusumoto, T. Kusama,
H. Brade, F. U. Schade, E. T. Rietschel and H. D. Flad,
Infect. Immun., 1992, 60, 3309–3314.
11 E. S. Van Amersfoort, T. J. C. Van Berkel and J. Kuiper, Clin. 31 C. Galanos, O. Luderitz, E. T. Rietschel, O. Westphal,
Microbiol. Rev., 2003, 16, 379–414.
H. Brade, L. Brade, M. Freudenberg, U. Schade, M. Imoto
12 E. T. Rietschel, T. Kirikae, F. U. Schade, U. Mamat,
and H. Yoshimura, FEBS, 1985, 148, 1–5.
G. Schmidt, H. Loppnow, A. J. Ulmer, U. Zahringer, U. Seydel, 32 Q. Wang and Z. Guo, Chem. Commun., 2009, 5536–5537.
F. Di Padova, M. Schreier and H. Brade, FASEB J., 1994, 8, 33 Y. Pan, P. Chefalo, N. Nagy, C. V. Harding and Z. Guo,
217–225.
J. Med. Chem., 2005, 48, 875–883.
13 A. G. Stover, J. D. S. Correia, J. T. Evans, C. W. Cluff, 34 Q. Wang, Z. Zhou, S. Tang and Z. Guo, ACS Chem. Biol.,
M. W. Elliott, E. W. Jeffery, D. A. Johnson, M. J. Lacy,
3244 | Org. Biomol. Chem., 2014, 12, 3238–3245
2012, 7, 235–240.
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
35 J. Wu and Z. Guo, Bioconjugate Chem., 2006, 17, 1537– 40 A. L. Gavin, N. Barnes, H. M. Dijstelbloem and
1544. P. M. Hogarth, J. Immunol., 1998, 160, 20–23.
36 T. Buskas, S. Ingale and G. J. Boons, Angew. Chem., Int. Ed., 41 D. H. Persing, R. N. Coler, M. J. Lacy, D. A. Johnson,
2005, 44, 5985–5988.
37 F. Estevez, A. Carr, L. Solorzano, O. Valiente, C. Mesa,
J. R. Baldridge, R. M. Hershberg and S. G. Reed, Trends
Microbiol., 2002, 10(Suppl.), S32–S37.
O. Barroso, G. V. Sierra and L. E. Fernandez, Vaccine, 1999, 42 Q. Wang and Z. Guo, ACS Med. Chem. Lett., 2011, 2, 373–
18, 190–197. 378.
38 Q. Wang, S. A. Ekanayaka, J. Wu, J. Zhang and Z. Guo, Bio- 43 R. M. Zinkernagel, S. Ehl, P. Aichele, S. Oehen, T. Kundig
conjugate Chem., 2008, 19, 2060–2068. and H. Hengartner, Immunol. Rev., 1997, 156, 199–209.
39 R. B. Markham, G. B. Pier and J. R. Schreiber, J. Immunol., 44 D. T. O’Hagan, M. L. MacKichan and M. Singh, Biomol.
1991, 146, 316–320.
Eng., 2011, 18, 69–85.
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 3238–3245 | 3245