Investigating the effects of a hydrolytically stable hapten and a Th1 adjuvant on heroin vaccine performance

Article abstract of DOI:10.1021/jm301262z

We challenged the performance of our previous heroin vaccine with a similar vaccine containing a more hydrolytically stable hapten analogue and a Th1 adjuvant (CpG ODN). Our results indicate that the elements of our previous vaccine are essential for its anti-heroin potency, i.e., a chemically labile hapten and an exclusively Th2 humoral response elicited by alum. Such design elements are critical for producing next-generation heroin vaccines.

Full text of DOI:10.1021/jm301262z

Brief Article
pubs.acs.org/jmc
Investigating the Effects of a Hydrolytically Stable Hapten and a Th1
Adjuvant on Heroin Vaccine Performance
Paul T. Bremer and Kim D. Janda*
Departments of Chemistry and Immunology, The Skaggs Institute for Chemical Biology, Worm Institute of Research and Medicine
(WIRM), The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States
S
* Supporting Information
ABSTRACT: We challenged the performance of our previous heroin vaccine with a similar vaccine containing a more
hydrolytically stable hapten analogue and a Th1 adjuvant (CpG ODN). Our results indicate that the elements of our previous
vaccine are essential for its anti-heroin potency, i.e., a chemically labile hapten and an exclusively Th2 humoral response elicited
by alum. Such design elements are critical for producing next-generation heroin vaccines.
INTRODUCTION
■
Heroin addiction is a neuropsychiatric disorder characterized by
compulsive administration of the powerful analgesic drug
heroin despite negative physical, mental, legal, and social
consequences. Worldwide, heroin addiction causes significant
detriment to society and facilitates the spread of blood-borne
pathogens such as HIV, HBV, and HCV.1 Traditional
pharmacotherapeutic treatment for heroin addiction consists
of small-molecule agonists/antagonists that blunt heroin
cravings and/or withdrawal symptoms. Although these small-
Figure 1. Heroin immunoconjugate structures.
molecule therapies are routinely employed to treat heroin
addiction in the clinic, they have many disadvantages such as
harmful side effects,2 demand for specialized infrastructure and
antibody titers specific for heroin and its deacetylated
personnel,3 high cost,4 and poor prevention of post-treatment
metabolites 6-acetylmorphine (6AM) and morphine. Addition-
drug relapse.5,6
ally, heroin analgesia and self-administration studies in
Immunopharmacotherapy (i.e., drugs of abuse vaccines) is a
potentially more attractive option for the treatment of heroin
addiction in comparison to pharmacotherapy. For the case at
hand, a heroin vaccine consists of a haptenic structure
mirroring the opioid scaffold, which is chemically linked to
an immunogenic carrier protein. When the hapten−protein
conjugate is injected, it elicits a humoral immune response
against heroin. Polyclonal antibodies in the periphery bind the
drug with a high degree of specificity, precluding entry into the
brain, thus blocking its psychological effects. Furthermore,
formulation of the immunoconjugate with an immunostimula-
tory adjuvant enhances the humoral response against the target
antigen. Therefore, a vaccine possessing an effective heroin-
mimetic hapten and the appropriate adjuvant has great
potential to effectively treat heroin addiction because in theory,
it can ablate the pharmacodynamical impact of a heroin dose.
Traditional treatments can only ameliorate symptoms caused
by withdrawal or prevent drug cravings but do nothing to
neutralize the drug once injected. Furthermore, drugs of abuse
vaccines have a low side effect profile7 and can protect against
drug relapse many months after immunization.8
vaccinated rats validated the in vivo efficacy of our heroin
vaccine.9 We hypothesized that the success of our vaccine is
attributed to its dynamic nature: the hapten is able to mimic
heroin metabolism, presenting epitopes similar to heroin, 6AM,
and morphine. In contrast, the deacetylated, more metabolically
static version of 7a (morphine-like hapten) elicited antibodies
with poor affinity for heroin and 6AM and displayed reduced in
vivo performance.9
Herein, we describe a more hydrolytically stable variation on
our previous heroin hapten in which the labile 3-acetate is
replaced with a robust 3-acetamide isostere (7b, Figure 1).
First, a vaccine embracing chemical epitope stability would test
our hypothesis that a “dynamic” hapten may be essential for
eliciting an anti-heroin humoral immune response. The 3-
acetamide would resist hydrolysis, thus preventing the usual
conversion to the deacetylated hapten. As a result, the antibody
affinity profile for heroin and its metabolites may be altered
drastically. Second, improved hapten stability may elevate anti-
heroin antibody titers. Such was the case for a study aimed at
generating a more potent anti-cocaine immune response:
diester to diamide substitution led to a more potent antibody
response to cocaine and showed impressive results in blunting
In a previous study from our laboratory, we identified a
heroin−KLH (keyhole limpet hemocyanin) hapten−protein
immunoconjugate (7a, Figure 1) which, in formulation with the
Th2 adjuvant alum (insoluble aluminum salts), generated high
Received: September 1, 2012
Published: November 7, 2012
© 2012 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
Scheme 1. Synthesis of 3-Acetamidoheroin
the effects of the drug in animal models.10 Third, our previous
hapten contained one critical liability involving its stability
design. Under the conditions in which it is formulated (pH 7.4,
PBS buffer, 25 °C), our heroin hapten in 7a has an estimated
97 h half-life.11 As a consequence, freezing or injection of the
immunoconjugate must occur immediately after preparation.
Conversion of one or both of the labile esters in our hapten to
amides (7b,c,d) would significantly increase vaccine half-life,
potentially permitting long-term storage.
Subsequent hydrolysis of 3 with hydroxylamine HCl unveiled
the aniline 4.17 Finally, exposure of 4 to acetyl bromide
accomplished concomitant desilylation and bisacetylation of the
3-amino and 6-alkoxy substituents, furnishing 5, the desired 3-
aza-analogue of heroin.18 Linker installment of 5 to hapten 6
and subsequent conjugation to KLH via sulfhydryl−maleimide
coupling were performed based on our previously established
protocols (Supporting Information and Scheme 2).9
We also sought to investigate the effect of a Th1 adjuvant
(CpG ODN 1826) on heroin vaccine performance. CpG
ODNs are well-studied adjuvants known to promote Th1
responses via stimulation of the innate immune receptor, TLR-
9. Although Th1 immunity is associated with cell-mediated
responses, it can concurrently manifest humoral responses. For
example, K-type (aka B-type) CpG ODNs such as ODN 1826
induce robust B cell activation, antibody production, dendritic
cell maturation, and TNF-α/IL-6 secretion in cell culture.12 In
vaccine trials, CpG ODN 1826 in combination with alum (Th2
adjuvant) has shown an amazing ability to promote IgG
antibody production against the target antigen (10−100-fold
greater titer compared to alum alone) in both mice13 and
humans.14 This improvement likely involves the ability of the
adjuvants working in tandem to noncompetitively promote IgG
isotypes associated with both Th1 and Th2 responses (IgG2a,b
and IgG1, respectively).13a,c Similarly, we applied this strategy
to our heroin vaccine by formulating both CpG ODN and alum
with our immunoconjugate. We hypothesize that activation of
both Th1 and Th2 humoral responses may be beneficial for
increasing heroin binding capacity over Th2 responses alone.
Scheme 2. Conjugation of Thiol Heroin Haptens to KLH
To compare the performance of the new hapten and
adjuvant with our previous vaccine, mice were immunized with
either hapten−protein conjugate 7b or 7a in formulation with
alum or alum + CpG ODN 1826. Vaccine efficacy was
measured by ELISA to assess anti-heroin antibody titers and
competitive ELISA to assess antibody affinity and specificity for
heroin and its metabolites.
Contrary to our hypothesis, ELISA results indicate a
significant negative effect of CpG ODN 1826 on titer levels
regardless of hapten structure (Table 1). Furthermore, the
adjuvant seems to have a noticeable effect on antibody
specificity and affinity for heroin and 6AM. ODN 1826
improved affinity for heroin and 6AM in the 7b vaccine group,
however, it caused marked reduction in 6AM affinity in the 7a
vaccine group.
We believe the CpG ODN 1826-mediated modulation of
anti-heroin antibody responses involves its well-documented
ability to induce Th1 polarization.12−14 The ODN adjuvant
possibly overpowered the Th2-inducing effects of alum. Given
the 3−4-fold lower titer for ODN treated groups (Table 1),
CpG ODN appears to reduce the overall humoral response
against the heroin immunoconjugate potentially in favor of cell-
mediated responses. In contrast to studies showing CpG ODN-
RESULTS AND DISCUSSION
■
To obtain the 3-acetamide analogue of our previous hapten in
7a, we first synthesized the 3-acetamide analogue of heroin
(Scheme 1). The 3-aminomorphine precursor has been
reported previously,15 however, we were not able reproduce
the synthesis and therefore developed a new, more efficient
route to the 3-aza-analogue (Scheme 1). Morphine sulfate
decahydrate was converted to 1 by selective triflation of the
phenol with N-phenyltriflamide. Protection of the hindered C-6
alcohol was accomplished with the highly electrophilic reagent
TBSOTf.16 The key step of the synthesis involved palladium-
catalyzed Buchwald amination of aryl triflate 2, resulting in the
replacement of the triflate to afford the imine intermediate 3.17
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a
Table 1. Anti-Heroin Antibody Titer, Specificity, and Affinity of Various Immunoconjugate-Adjuvant Formulations
OD50 (μM)
vaccine
7a + alum
IgG titer
IgG1 titer
IgG2a titer
1/2a ratio
heroin
6AM
morphine
11800 2000 a
3840 890 a
13400 1700 b
4750 750 b
21800 3400
4590 70
1290 40
480 54
16.9 g
9.61 g
6.46
3.44 0.33 d
5.07 1.48
<0.391 e,f
42.9 9.5 e
31.8 17.1 f
2.22 0.58
>400
>400
>800
>800
7a+ alum + ODN
7b+ alum
14600 2600
6110 680
2260 180
1180 60
42.9 9.5 c,d
12.3 3.9 c
7b+ alum + ODN
5.18
a
All values are reported as means SEMs. Differences between values with matching letters are statistically significant (P < 0.05).
mediated promotion of humoral responses against target
proteins,12,13 our results indicate that this adjuvant and
potentially all TLR-9 agonists are not well suited for vaccines
against small molecules, especially in the presence of alum.
However, optimizing the dosage,19 vaccination schedule, and
molecular target of Th1 adjuvants may yield improvement in
future studies.
An in-depth analysis of Th1 vs Th2 antibody isotypes
revealed that CpG ODN shifted the ratio of IgG1/IgG2a titer
to increase IgG2a (Th1) relative to IgG1 (Th2) in the 7a
groups (Table 1). A similar trend is observed for the 7b groups.
As expected, the ODN adjuvant favored the generation of Th1-
associated antibodies although Th2-associated antibodies
predominated. The shift in isotype ratio may be correlated
with the shift in antibody specificities and affinities for heroin
and 6AM (Table 1). In a broader sense, this result provides
intriguing evidence that antibody isotype influences affinity and
specificity for small molecules: a burgeoning theory that links
the binding behavior of the antibody variable domain with the
structure of the constant domain.20
In addition to adjuvant effects, hapten structure appears to
greatly influence antibody character in accordance with our
original hypothesis. Although both haptens appear equally
immunogenic (similar titer levels, Table 1), 6AM affinity
decreases significantly (∼1000-fold) when the 3-acetamide is
incorporated into the hapten. A likely explanation involves the
fact that the acetamide cannot be hydrolyzed, precluding
conversion to a 6AM- or morphine-like epitope, which both
contain the C-3 alcohol. In contrast, 12-fold lower heroin
affinities in the 7b vaccine cannot be explained in the same
manner because heroin is the parent compound in the
metabolism pathway. The lower heroin affinities are likely a
result of the 3-acetamide hapten presenting a slightly altered
epitope from the native heroin hapten (3-acetate) in 7a.
Although the absolute OD50 values for heroin and 6AM for 7a
compare well with our previous study, morphine values do not,
which may be attributable to differences between the host
animals (mice herein vs rats previously) and their carbox-
ylesterase profiles.21
acetylmorphine 5 directly converts to 3-acetamidomorphine 8
(Supporting Information). The results obtained from this study
can be extrapolated to immunoconjugates 7a and 7b; the
stability study indicates that we have in fact created a more
stable heroin vaccine via 3-acetate to 3-acetamide substitution.
We believe our half-life results in buffer present a suitable
comparison of relative hapten stabilities which likely hold true
in the various biological environments encountered by the
vaccine in vivo. Additionally, hydrolysis of 7b hapten directly to
a 3-acetamidomorphine-like structure (dissimilar to both 6AM
and morphine) explains the poor antibody affinity for 6AM,
further demonstrating the need for chemical lability at the 3-
position.
CONCLUSION
■
In this paper, we have made significant progress in under-
standing the necessary design elements of a successful heroin
vaccine. In testing the hydrolytically stable 3-acetamide
immunoconjugate (7b), we have determined that the
metabolically dynamic nature of our previous immunoconju-
gate (7a) appears to be essential for generating antibodies with
high specificity and affinity for heroin and 6AM, the two key
neutralization targets for blunting the addictive effects of
heroin.9,22 Our findings demonstrate proof-of-concept that
blocking hapten metabolism through rational hapten design
directly translates to antibody affinity for cognate, upstream
drug metabolites. The overall outcome of our study indicates a
clear direction for a next-generation heroin vaccine.
Although our 3-acetamide vaccine displayed enhanced
resistance to hydrolysis, its significantly reduced antibody
affinity for 6AM limits its therapeutic potential as a heroin
vaccine; however, development of a vaccine with the 6-
acetamidoheroin hapten (7c, Figure 1) may be more successful.
In contrast to 7b, 7c would present a labile heroin-like and a
highly stable 6AM-like epitope to the immune system that may
boost antibody affinity for 6AM. Synthesis of 6-acetamido-
opioid analogues is well documented and consists of oxidation
of the C-6 alcohol to the ketone followed by reductive
amination and subsequent acetylation.23 Applying the same
chemistry to 5 (Scheme 1) would result in diacetamidoheroin
which can be used to create conjugate 7d. This conjugate may
be useful because it would possess maximum stability resulting
in exclusive presentation of a heroin-like epitope in vivo.
To our knowledge, we are the first group to outfit a drugs of
abuse vaccine with a CpG ODN adjuvant.24 Although the
adjuvant reduced titer levels, it provides insight into the
immunochemical mechanisms that underlie active immuniza-
tion against small-molecule drugs. Drugs of abuse vaccines are
fundamentally different than vaccines against polyvalent
bacteria or viral pathogens because they induce a robust
humoral response that only serves to neutralize drug molecules.
Cell-mediated responses to the drug are likely unnecessary if
Lastly, a mass spectrometric assay has confirmed the
enhanced stability of our 3-acetamide hapten. In comparison
to heroin, 5 has a noticeably longer half-life because the labile
ester is replaced with an amide (Table 2). However, the 6-
acetate of 5 is still prone to hydrolysis and therefore possesses a
half-life similar to 6AM. Under basic conditions, 3-acetamido-6-
Table 2. Half-Lives (h) of Opiates in Buffer at 25 °C
b
b
pH
5
6AM
heroin
a
a
7.4
9.0
stable
stable
45.9
96.8 1.3
5.9
43.8 0.4
a
b
Half-life >2 weeks. Literature data.11
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(Eurofins, 5′-TCCATGACGTTCCTGACGTT-3′) was added to the
vaccine formulation (40 μg per mouse per injection). Serum was
collected at days 21 and 42. Titers were higher in the 42 day sera
which was used to obtain the reported data. No anti-heroin antibodies
were detected in the sera of KLH control mice.
Statistics. Microsoft Excel 2007 was used for data management
and for linear regression analysis of calibration curves for the mass spec
stability assay. GraphPad Prism version 6 was used for calculating
means SEMs and for performing nonlinear regression analyses and t
tests for statistical significance. ELISA OD50 values were determined
by plotting log(dilution) vs absorbance and then applying the “one
site-fit log IC50” nonlinear regression. Half-life was determined by
applying the “one phase decay” exponential regression to a plot of time
vs %[5] relative to [5 + 8].
not deleterious to vaccine performance. Our vaccine study
suggests TLR-9 plays a role in the immunochemical
mechanisms behind drugs of abuse vaccine action due to the
ability of CpG ODN 1826 to significantly modulate vaccine
performance. CpG ODN 1826 activation of TLR-9 may favor
Th1 cell-mediated responses, causing a reduction in alum-
mediated Th2 humoral responses to heroin haptens. Also, CpG
ODN-induced isotype switching of IgGs may alter antibody
specificity and affinity profiles for heroin-like compounds; a
potential example of IgG isotype switching influencing the
small molecule binding profile of antibodies.
Our investigation of CpG ODN 1826 adjuvant on heroin
vaccine efficacy highlights the importance of adjuvant
formulation with drugs of abuse vaccines. Although haptenic
variation has been heavily investigated to optimize therapeutic
potency, thorough study of adjuvants in drugs of abuse vaccines
has received less attention. The ability of adjuvants to polarize
the immune response plays a critical role in fine-tuning humoral
responses to maximize drug molecule neutralization. In our
case, TLR-9 stimulation appears to greatly downregulate anti-
heroin humoral responses relative to alum, thus begging the
question of how other TLRs influence immune response to
drugs of abuse vaccines. Clearly, proper adjuvant selection and
formulation is essential for the future success of drugs of abuse
vaccines in combating drug addiction and will be reported in
due course.
ASSOCIATED CONTENT
■
S
* Supporting Information
Compound characterization spectra (1H NMR, 13C NMR, LC-
UV) and experimental procedures. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: (858) 784-2515. Fax: (858) 784-2595. E-mail:
kdjanda@scripps.edu.
■
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
EXPERIMENTAL SECTION
Synthetic preparation of haptens, ELISAs, and mass spectrometric
assay details are presented in the Supporting Information.
■
Notes
The authors declare no competing financial interest.
Hapten Conjugation to KLH (Scheme 2). Trityl protected
haptens 6a and 6b (4 mg, 5.3 μmol) were deprotected to thiols 6aSH
and 6bSH by stirring at room temperature in a solution of DCM (0.5
mL), TIPS (100 μL), and TFA (100 μL) for 15 min. The solvent was
blown off with nitrogen, and the residue was placed under vacuum for
15 min. Triethylamine (50 μL) and DCM (1 mL) were added to the
residue, which was then purified by silica preparative TLC [15%
MeOH/DCM to afford the thiol 6a/bSH as a clear residue (2.0−2.2
mg, ∼75%)]. To a pH 7.4 PBS solution of KLH (10 mg/mL, 350 μL)
was added sulfo-GMBS in conjugation buffer (CB) from Thermo
Pierce (4.3 mM, 175 μL). The solution was stirred for 1 h at room
temperature and dialyzed at 4 °C [4 h × 5 (50 mL pH 7.4 PBS)].
Immediately following deprotection, 6a/bSH (2.0−2.2 mg) was
dissolved in a 20% DMSO/CB solution (250 μL) and added to the
maleimide-activated KLH solution. The solution was stirred for 3 h at
room temperature and dialyzed at 4 °C [4 h × 3 (50 mL pH 7.4
PBS)]. Solutions of immunoconjugates 7a/b were diluted 1:2 with
glycerol and stored at −20 °C until injection. The final
immunoconjugate concentrations of 7a and 7b were equal (1.5−1.8
mg/mL of protein in 1.8 mL buffer) as determined by both NanoDrop
and BCA assay (Thermo Pierce). During the conjugation, the hapten
concentration was monitored by an Ellman assay using a protocol
from Thermo Pierce. Assay results reveal equal degrees of haptenation
between both KLH immunoconjugates 7a and 7b (0.7−0.9 mmol
hapten/mg KLH).
Lastly, BSA conjugates analogous to 7a and 7b were made in the
same manner to perform MALDI-ToF mass spectrometric analysis
and ELISAs. Mass spectometric results also show an equal degree of
haptenation between the two BSA conjugates of five hapten copies per
BSA protein (Supporting Information).
Immunization. Five groups of five female Swiss Webster mice
(Taconic Farms; 6−8 weeks old; 25−30 g) were immunized with
KLH (control), 7a + alum, 7a + alum + ODN, 7b + alum, or 7b +
alum + ODN. Immunoconjugates were administered ip in doses of
100 μg per mouse on days 0, 14, and 28. All conjugates were
formulated in a 1:1 v/v mixture of alum (Thermo Pierce) immediately
before injection. In the ODN treated groups, CpG ODN 1826
ACKNOWLEDGMENTS
■
We thank Neil G. Stowe for his support in passing on the
heroin vaccine project. This work was funded by the Skaggs
Institute for Chemical Biology at the Scripps Research Institute
and by the National Institutes of Health under grant no. R01-
DA026625.
ABBREVIATIONS USED
■
KLH, keyhole limpet hemocyanin; 6AM, 6-acetylmorphine;
ODN, oligodeoxynucleotide; GMBS, N-(γ-maleimidobutyry-
loxy) sulfosuccinimide ester)
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