Lipid tucaresol as an adjuvant for methamphetamine vaccine development

Article abstract of DOI:10.1039/c4cc00682h

The immunopotentiator tucaresol was modified for incorporation into liposomes, where it was found to be a superior adjuvant to MPLA for vaccination against methamphetamine. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc00682h

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Lipid tucaresol as an adjuvant for
methamphetamine vaccine development†
Cite this: Chem. Commun., 2014,
50, 4079
K. C. Collins,a J. E. Schlosburg,ab J. W. Lockner,a P. T. Bremer,a B. A. Ellisa and
K. D. Janda*a
Received 26th January 2014,
Accepted 28th February 2014
DOI: 10.1039/c4cc00682h
www.rsc.org/chemcomm
The immunopotentiator tucaresol was modified for incorporation IgM to IgG, effecting a specific and long-lasting immune response.
into liposomes, where it was found to be a superior adjuvant to Vaccines against nicotine and cocaine have reached clinical trials,
MPLA for vaccination against methamphetamine.
but candidates against methamphetamine are still in early stages
of development.10 One area of vaccine optimisation is the choice of
Methamphetamine abuse has been an increasing global problem adjuvant, which is used to enhance the local immune response by
over the last few decades,1 and was estimated to cost over $23 billion increasing (local) inflammation, stimulating antigen presenting
in 2005 in the US alone.2 Methamphetamine is highly addictive, cells and acting as a depot.11 Alum (aluminium salts) has been the
flooding the brain with multiple neurotransmitters, including historically dominant adjuvant, but alternatives are being pursued
dopamine, serotonin and norepinephrine, causing a euphoric in an effort to improve safety, increase the strength of the immune
high.3 Repeated exposure depletes neurotransmitter levels and response and to access alternative immune response profiles.12
damages the corresponding transporter systems,4 resulting in severe Liposomes have been explored as vaccine delivery systems since
withdrawal on attempted abstinence, and consequently a gravely 197413 and are currently at the forefront of vaccine research due to
high relapse rate.5 Furthermore, the complex neurochemistry their ability to safely deliver both antigen and adjuvant in a
behind the drug’s psychoactive effects makes development of versatile and readily-optimisable manner at relatively low cost.
treatments difficult; there are no approved medications for
Monophosphoryl lipid A (MPLA) is the only non-alum adjuvant
methamphetamine addiction.6,7 The only current treatments are approved for use (in conjunction with alum) in both the US and
behavioural therapies,8 which require a significant support network Europe14,15 A detoxified derivative of bacterial lipopolysaccharide
that addicts frequently lack, and which have demonstrated only (LPS), MPLA is believed to enhance the immune system via a
limited improvement in long-term abstinence rates.9 One attractive combination of mechanisms including agonism of toll-like
therapeutic approach in development is active immunisation receptor 4 (TLR4), which invokes a signal cascade that results
against methamphetamine; a successful vaccine would aid in the production of proinflammatory cytokines16 and antigen-
abstinence by sequestering the drug in the event of a relapse, specific effector CD4+ and memory CD8+ T cells.17 Synthetic
minimising its pharmacological effects.
MPLA (also termed phosphorylated hexaacyl disaccharide,
Small molecules like methamphetamine are invisible to the PHAD or glycopyranoside lipid A, GLA) has also more recently
immune system, traditionally requiring conjugation to a carrier been investigated as an alternative to the multi-component,
to make them immunogenic; the peptidic nature of this carrier potentially heterogeneous, bacteria-derived MPLA.18 Direct
results in presentation of the MHC class II–antigen complex to comparison has shown enhanced results using the synthetic
the immune system, initiating antibody isotype switching from version,19 which being homogeneous, allows for precise
control over the vaccine components. MPLA has been incorporated
into liposomes (L(MPLA)) where it has shown stronger
immunostimulation than alum, and synthetic L(MPLA) has been
a Departments of Chemistry, Immunology and Microbial Science,
The Skaggs Institute for Chemical Biology, Worm Institute of Research and
applied to drugs of abuse vaccines, where it has successfully
elicited high antibody titres against heroin.20
Medicine (WIRM), The Scripps Research Institute, 10550 North Torrey Pines Road,
La Jolla, California 92037, USA. E-mail: kdjanda@scripps.edu;
Fax: +1-(858)-784-2595; Tel: +1-(858)-784-2515
b Committee on Neurobiology of Addictive Disorders, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, California 92037, USA
† Electronic supplementary information (ESI) available: Details of synthetic
procedures, detailed ELISA and RIA data and NMR spectra. See DOI: 10.1039/
c4cc00682h
Another class of molecular adjuvant under investigation in clinical
trials are the Quillaja saponins, which effect immunostimulation by
providing T cells with direct costimulatory signals; the aldehyde
moieties they contain are believed to mimic carbonyl groups on the
surface of antigen-presenting cells (APCs), forming Schiff bases with
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free lysine residues on the surface of T cells.21 MPLA and
saponins are complex small molecules and herein we propose
that a readily synthetically accessible adjuvant, tucaresol, may
be a suitable substitute. Tucaresol is an orally bioavailable
aldehyde-containing immunopotentiator, whose application
in vaccines has been primarily limited to systemic use to
enhance DNA-based vaccines,22 but when used as a local
adjuvant it has demonstrated enhanced Th cell priming compared
to both alum and saponin Quil A.23 Tucaresol has been shown to
elicit both cellular and humoural responses,22 with characteristics of
both Th 1 and Th 2-type immunity;24 we have proposed that this
mixed response is ideal for drugs of abuse vaccines.25 Despite
tucaresol being identified as an immunopotentiatory agent two
decades ago, its use in vaccines has not flourished. Although
tucaresol stimulates T cells, it has not been directly associated with
the antigen as part of the vaccine design, and is usually administered
Scheme 1 Synthesis of lipid tucaresol, LT1 and MH6t-CO2H, 2.
Having chosen our target adjuvant and hapten, lipid tucaresol
orally or in a separate injection. We envisaged that incorporation of LT1 was therefore synthesised in two steps from 4-(bromomethyl)-
tucaresol into liposomes would enhance immunopotentiation by benzoic acid via esterification with 1-hexadecanol, followed by
recruiting the liposomes to the T cells, promoting uptake of the etherification with 2,6-dihydroxybenzaldehyde (Scheme 1a). MH6t-
liposomes and thus the associated antigen. Modification of tucaresol CO2H, 2 was synthesised from methamphetamine via alkylation of
via lipidation would generate an amphiphilic molecule capable of the amine followed by saponification of the ester (Scheme 1b).
being incorporated into the lipid bilayer of liposomes. We proposed
that esterification of the acid would be a facile method to achieve diphtheria toxoid (DT) and BSA, using standard acid activation
this and here provide the first reported example of a lipid analogue (see ESI†). The DT conjugate, MH6t(CO2H)–DT (25 mL, 1 mg mLÀ1
MH6t-CO2H, 2 was then conjugated to two carrier proteins,
,
of tucaresol (lipid tucaresol, LT1, Fig. 1).
hapten density of 13.3), was mixed with liposomes containing
either MPLA or LT1 (25 mL) with the following composition:
DMPC (90 mM), cholesterol (75 mM), DMPG (10 mM) and
either MPLA (0.454 mM, 20 mg per injection) or tucaresol
(0.454 mM, 5.6 mg per injection). The resulting vaccines,
MH6t(CO2H)–DT + [L(MPLA)] and MH6t(CO2H)DT + [L(LT1)]
were used to immunise groups of six Swiss Webster mice
(6–8 weeks old) via subcutaneous injection on days 0, 14, and
35. Serum was collected via tail bleed on days 21 and 42, and
via cardiac puncture on day 63, and production of anti-
methamphetamine antibodies was evaluated by ELISA using
MH6t(CO2H)–BSA coated plates (hapten density of 30.3). Anti-
body affinities and concentrations were determined by radio-
immunoassay (RIA, Fig. 3).
Fig. 1 Structures of tucaresol and lipid tucaresol, LT1.
Another key variable in drugs of abuse vaccine design is the
choice of hapten. Our best candidate for vaccination against
methamphetamine is MH6-SH (Fig. 2).26,27 This hapten
requires thiol conjugation to the appropriately-activated carrier
protein since it contains a secondary amine that would be
incompatible with conjugation using activated carboxylic acids.
We have found acid hapten conjugations to give more robust,
efficient conjugation than their thiol counterparts, with the added
benefit that the hapten precursors are stable upon storage.
We therefore designed MH6t-CO2H (2, Fig. 2) as a new hapten,
proposing that switching from a secondary to a tertiary amine
should not have a significant impact on antibody production;
elsewhere it has been shown that anti-methamphetamine anti-
bodies can still be generated despite the larger perturbation of
derivatising this amine to a tertiary amide.28
Both liposomal vaccines showed
a similar immune
response, with tucaresol appearing to be the superior adjuvant
in this case; L(MPLA) and L(LT1) generate similar antibody
concentrations (19 and 14 mg mLÀ1, respectively), but the
specificity of the L(LT1)-induced antibodies is higher, with a
Kd value of 134 nM (compared to 233 nM induced by
[L(MPLA)]). This value is also on par with that obtained in a
previous study using secondary amine hapten MH6(SH)-KLH
with SASs under a similar vaccination schedule (130 nM at
42 days).26 This suggests that the presence of the extra methyl
group in the MH6t hapten does not substantially impact anti-
body recognition of methamphetamine. However, the concen-
tration of the antibodies elicited by MH6t(CO2H)–DT + [L(LT1)]
(14 mg mLÀ1 at 63 days) is lower than that of MH6(SH)-KLH +
SASs (108 mg mLÀ1 at 42 days). The antibody concentration
dropped throughout the study of MH6(SH)-KLH + SASs, and
given that our bleed was performed at a later time point, this
may account for some of the discrepancy. Analysis of the ELISA
Fig. 2 Structures of MH6-SH and MH6t-CO2H, 2.
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Notes and references
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Fig. 3 Anti-methamphetamine antibody titres, affinities and concentrations
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In summary, we have synthesised the first lipid analogue of
tucaresol, LT1, and shown that it is capable of eliciting an
immune response against methamphetamine, demonstrating
superior liposomal adjuvancy to MPLA and having great
potential for future development.
This work was funded by the National Institute on Drug
Abuse grant nos. DA026625 and DA08590. This is manuscript
#26080 from The Scripps Research Institute.
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