Development of a Clinically Viable Heroin Vaccine

Article abstract of DOI:10.1021/jacs.7b03334

Heroin is a highly abused opioid and incurs a significant detriment to society worldwide. In an effort to expand the limited pharmacotherapy options for opioid use disorders, a heroin conjugate vaccine was developed through comprehensive evaluation of hap

Full text of DOI:10.1021/jacs.7b03334

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Article
Development of a Clinically-Viable Heroin Vaccine
Paul T. Bremer, Joel E. Schlosburg, Matthew L Banks,
Floyd F. Steele, Bin Zhou, Justin L. Poklis, and Kim D Janda
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 02 Jun 2017
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Development of a Clinically-Viable Heroin Vaccine
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Paul T. Bremer,1 Joel E. Schlosburg,2 Matthew L. Banks,2 Floyd. F. Steele,2 Bin Zhou,1 Justin L. Poklis,2 Kim
D. Janda1*
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1Departments of Chemistry and Immunology, The Skaggs Institute for Chemical Biology, Worm Institute of
Research and Medicine (WIRM), The Scripps Research Institute, 10550 N Torrey Pines Rd, La Jolla, CA
92037, USA
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2 Department of Pharmacology and Toxicology, Virginia Commonwealth University, 410 N 12th St, Richmond,
VA 23298, USA
Abstract
Heroin is a highly abused opioid and incurs a significant detriment to society worldwide. In an effort to
expand the limited pharmacotherapy options for opioid use disorders, a heroin conjugate vaccine was developed
through comprehensive evaluation of hapten structure, carrier protein, adjuvant and dosing. Immunization of
mice with an optimized heroinꢀtetanus toxoid (TT) conjugate formulated with adjuvants alum and CpG
oligodeoxynucleotide (ODN) generated heroin ‘immunoantagonism’, reducing heroin potency by >15ꢀfold.
Moreover, the vaccine effects proved to be durable, persisting for over eight months. The lead vaccine was
effective in rhesus monkeys, generating significant and sustained antiꢀdrug IgG titers in each subject.
Characterization of both mouse and monkey antiꢀheroin antibodies by surface plasmon resonance (SPR)
revealed low nanomolar antiserum affinity for the key heroin metabolite, 6ꢀacetylmorphine (6AM), with
minimal cross reactivity to clinicallyꢀused opioids. Following a series of heroin challenges over six months in
vaccinated monkeys, drugꢀsequestering antibodies caused marked attenuation of heroin potency (>4ꢀfold) in a
scheduleꢀcontrolled responding (SCR) behavioral assay. Overall, these preclinical results provide an empirical
foundation supporting the further evaluation and potential clinical utility of an effective heroin vaccine in
treating opioid use disorders.
Introduction
Heroin, a semisynthetic opioid, and its parent natural product, morphine, are among the longest known and
commonly abused psychoactive drugs. Heroin is a prodrug1 that readily crosses the bloodꢀbrain barrier while
quickly deacetylating to 6ꢀacetyl morphine (6AM) and then more slowly to morphine (Figure 1).2ꢀ3 These two
metabolites agonize brain muꢀopioid receptors (MOR) to produce heroin’s abuseꢀrelated euphoric and
reinforcing effects.4ꢀ6 Moreover, the robust analgesic effects of opioids have led to their extensive clinical use as
prescription painkillers such as OxyContin (oxycodone) and Vicodin (hydrocodone); however, these opioids are
also routinely abused and can act as “gateway drugs” to heroin.7ꢀ8 Persistent opioid abuse leads to a
neuropsychiatric disorder, i.e. opioid use disorder, characterized by compulsive opioid administration despite
the negative physical, mental, legal and social consequences of prolonged use.
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Figure 1. Structures of heroin and its key metabolites which act as prodrugs and/or MOR agonists.
Currently in the United States, opioid abuse has reached epidemic levels. The number of people who have
used heroin in the past 10 years has doubled from 379,000 in 2005 to 828,000 in 2015,9ꢀ10 and heroin
expenditures have grown steadily to an estimated $27 billion (2010) on drug purchases alone.11 The wideꢀ
spread prevalence of heroin abuse is a significant cost to users and to society as a whole (an estimated total of
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$22 billion in the US).12ꢀ13 Other negative impacts of heroin abuse include HIV or HCV infection, for which
injection drug users remain at the highest risk.14 While prescription opioids combined are involved in the most
drugꢀrelated deaths in the US, compared to any one single drug, heroin is responsible for at least twice as many
deaths.15
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Abuse of prescription opioids may be mitigated by tightening regulations or by introducing antiꢀabuse
technology during manufacturing. On the other hand, heroin and other synthetic opioids, e.g. acetyl fentanyl,
are produced and distributed illegally; therefore, great measures must be taken to curb opioid use disorders.
Current treatment options for opioid use disorders include opioid replacement therapy utilizing methadone or
buprenorphine as MOR agonists to reduce opioid withdrawal symptoms and maintain heroin abstinence.16ꢀ17
Opioid antagonists naloxone and naltrexone (NTX) are other treatment options, FDAꢀapproved for opioid
overdose and dependence, respectively.18ꢀ19 Pharmacological intervention for heroin abuse has proven to be
effective but has a number of drawbacks including high cost of inꢀpatient rehab,12ꢀ13 undesirable effects,20ꢀ21 and
relapse potential following therapy.13, 22ꢀ23
Humanity has benefited from vaccines for more than two centuries, and of all the biomedical achievements,
immunization for the prevention of infectious diseases ranks highly. The first attempt at translating vaccination
to reduce the abuse of psychoactive substances was reported in the early 70s when a conjugate vaccine
containing a morphineꢀlike hapten was tested in a single rhesus monkey.24 However, this work was not followed
up due to the emergence of pharmacotherapies for opioid use disorders, e.g. methadone, which at the time,
appeared more promising.25 Drug conjugate vaccine research reꢀemerged in the midꢀ90s, and focused on
cocaine26 and nicotine.27 Unfortunately, multiple failures of both cocaine and nicotine vaccines in human trials
have called into question the clinical value of vaccination for treating substance use disorders.28ꢀ31 Potential
problems of these vaccines include poor hapten design32 and adjuvant selection. Moreover, these vaccines
lacked rigorous preclinical development, as they have not demonstrated the ability to block a wide range of drug
doses in multiple behavioral procedures. Failure to address and ultimately solve these problems has hampered
progress in the drugꢀvaccine field.
The principle design elements behind drug abuse vaccines include a hapten (Bꢀcell epitope), highly
congruent in structure to the target drug, and an immunogenic carrier protein (Tꢀcell epitope) such as tetanus
toxoid (TT) (Figure 2). Immunization of the haptenꢀprotein conjugate formulated with adjuvants e.g. alum and
CpG oligodeoxynucleotide (ODN), triggers an adaptive immune response against the drugꢀlike hapten (Figure
2). Subsequently, when the vaccinated subject receives a drug dose, available polyclonal IgG antibodies in the
periphery bind the drug with a high degree of affinity and specificity, precluding drug entry to the brain (Figure
2). Moreover, the vaccine significantly attenuates the pharmacodynamics of the drug dose without modulating
receptors in the brain. The unique mechanism of action of the drugꢀconjugate vaccines may offer many
advantages for treating substance use disorders such as the potential for reduced side effects,33 convenient and
lowꢀcost administration, and longꢀterm efficacy.34
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Figure 2. Simplified schematic of heroin conjugate immunological pathway and mechanism of action. Upon vaccination, the
conjugate is taken up by dendritic cells (DCs), processed and displayed on the major histocompatibility complex class II (MHCII) as a
haptenated peptide for activation of helper Tꢀcells (Thꢀcells) via the Tꢀcell receptor (TCR). Bꢀcells which have encountered the
conjugate via their Bꢀcell receptors (BCR) are stimulated by activated Thꢀcells. Adjuvants alum and CpG ODN enhance the vaccine
response through NLRP3 inflammasome and TLR9 signaling, respectively. Memory Bꢀcells and plasma cells produce high affinity
antiꢀ6AM IgG antibodies which bind to an administered heroin dose (converted to 6AM) in the periphery, thus mitigating drug effects
in the brain.
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We envisioned the need for a comprehensive series of preclinical experiments wherein every component of
the vaccine would be scrutinized, i.e. hapten, carrier and adjuvant. Moreover, the cornerstone of our
development process involved the quantitative evaluation of each vaccine iteration in a rodent antinociception
assay. Thus, full heroin doseꢀresponse curves were generated to compare ED50 values of vaccinated and nonꢀ
vaccinated groups of rodents, providing a direct measure of the ‘immunoantagonistic’ capacity of the vaccine.35
A firstꢀgeneration heroin vaccine produced heroin ED50 ratios of 4ꢀ5 in both mice and rats,36 blocking the
effects of heroin in a series of behavioral models.37 In this work, a significant leap forward was made in
redesigning the vaccine to achieve a greater than 15ꢀfold heroin ED50 shift in rodents, warranting further
investigation in a primate behavioral model. The translational potential of the heroin vaccine was confirmed for
the first time in rhesus monkeys, supported by statistical analysis in n = 4 subjects.
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Results
Vaccine optimization: hapten, carrier and adjuvant
As depicted in Scheme 1, preparation of heroin haptens was accomplished by first demethylating heroin via
Olofson’s procedure.38 Reductive amination of Bocꢀprotected 4ꢀaminobutanal followed by TFA deprotection
afforded the key intermediate 1 as previously described.36 Amide coupling(s) followed by trityl or tꢀbutyl ester
deprotections yielded the first generation thiol hapten (5, HerSH)36 or novel second generation carboxylic acid
haptens (2-4). Thiol and carboxylic acid haptens were coupled to surface lysines of carrier proteins via thiolꢀ
maleimide or amide couplings, respectively. At a 1:1 w/w ratio of hapten to protein, carboxylate haptens
showed higher hapten loading according to MALDIꢀToF analysis (Table S1). Additional advantages to
carboxylate conjugations included resistance to oxidation and a oneꢀpot coupling procedure. In contrast, thiols
can form disulfides and require preparation of maleimideꢀloaded proteins prior to conjugation, i.e., a twoꢀpot
procedure.
Scheme 1. Synthesis and conjugation of heroin haptens.
Three of the most commonly used carrier proteins keyhole limpet hemocyanin (KLH), diphtheria toxoid
(DT) and tetanus toxoid (TT) were investigated in the context of the heroin conjugate vaccine. In comparing the
proteins as HerSH conjugates, TT, which is routinely used in humanꢀapproved tetanus vaccines, showed the
best performance in attenuating heroinꢀinduced antinociception in mice (Figure 3A) and produced the greatest
antiꢀhapten titers (Figure S2A). Bioconjugation methods and linker structures were also compared through the
testing of TT conjugates of HerSH (5), HerCOOH (2) and HerdBA (4), and the HerCOOH hapten demonstrated
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the greatest efficacy (Figure 3B, S2B). While the amide coupling method appeared to generate more efficacious
conjugate compared to thiolꢀmaleimide coupling, this benefit appeared to be erased by the presence of the diꢀ
betaꢀalanine (dBA) linker found in the HerdBA hapten. The effect of the 3ꢀacetyl group was probed through a
6AMꢀlike hapten (6AMCOOH, 3), because 6AM is the main mediator of heroin psychoactivity.4ꢀ6 Behavioral
results indicated that the 6AM and the heroin haptens were comparable in efficacy (Figure 3C). ELISA results
corroborated the behavior because sera from both groups bound both 6AM and heroin haptens to a similar
degree; however, the 6AM conjugate elicited antibodies with a reduced capacity to bind the heroin hapten
(Figure S2C).
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In continuing studies with the HerCOOHꢀTT conjugate, the effect of conjugate dosing and adjuvant was
explored, using FDAꢀapproved alum adjuvant (Al(OH)3) alone as a benchmark. Conjugate dosing demonstrated
a positive effect on titer levels and mitigation of heroin antinociception (Figure S2D, 3D). Addition of CpG
ODN 1826, a Tollꢀlike receptor (TLR) 9 agonist, enhanced vaccine potency of HerCOOHꢀTT (Figure 3E) as
previously shown for HerSHꢀKLH.35 Without alum, CpG ODN was not effective (data not shown). When both
conjugate and CpG ODN doses were doubled to 100 ꢁg and 60 ꢁg respectively, heroin ‘immunoantagonistic’
capacity and antiꢀhapten titers increased dramatically (Figure 3E, S2E). A second DNAꢀbased
immunostimulatory adjuvant, 2’3’ꢀcyclic guanosine monophosphateꢀadenosine monophosphate (cGAMP)39 was
also evaluated. Although both cGAMP and CpG ODN target the innate immune system, they activate different
receptors and pathways via STING and TLR9, respectively. A mild vaccine improvement was noted with low
dose cGAMP, however, this effect was not present at a higher cGAMP dose (Figure 3E). The outcome of these
optimization studies was the identification of a lead vaccine formulation containing HerCOOHꢀTT, CpG ODN
and alum.
Figure 3. Optimization of heroin immunoconjugate and vaccine formulation. (A) Comparison of carrier proteins with HerSH
hapten. ##P < 0.01, ###P < 0.001, **P < 0.01 versus KLH. (B) Evaluation of heroin haptens as TT immunoconjugates. ###P < 0.001,
***P < 0.001 versus HerSHꢀTT. (C) Comparison study of 6AMCOOH vs. HerCOOH haptens as TT conjugates. (D) Doseꢀ
dependency of HerCOOHꢀTT immunoconjugate on vaccine efficacy. ##P < 0.01, ###P < 0.001, *P < 0.05 versus 10 µg dose. No CpG
ODN was used. (E) Adjuvant effects of CpG ODN 1826 and cGAMP on vaccine efficacy. Doses: 4 µg CpG (Low CpG), 30 µg CpG
(High CpG), 60 µg CpG + 100 µg HerCOOHꢀTT (2X dose), 1.3 µg (Low cGAMP), 10 µg (High cGAMP). #P < 0.05, ##P < 0.01, **P
< 0.01, ***P < 0.001 versus alum only. Vaccine formulations for all panels contained 0.75 mg alum, 30 µg CpG ODN 1826 and 50 µg
HerCOOHꢀTT unless otherwise noted and were administered to n = 6 mice i.p. at wk 0, 2 and 4. For all panels, the mean heroin ED50
± SEM (determined via cumulative s.c. dosing) is shown and statistics were evaluated by a oneꢀway ANOVA with Tukey’s postꢀhoc
test. Testing was performed at wk 6 and significance is denoted by (#) for tail immersion and (*) for hot plate. Foldꢀshifts in ED50
versus n = 6 nonꢀvaccinated control mice are reported above each set of data points.
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Long-term duration of lead vaccine efficacy
In order to test the durability of the optimized heroin vaccine, an extended vaccination study was performed
over a 37ꢀweek period in mice. After an initial immunization, peak antiꢀheroin titers were observed by ELISA,
causing significant potency shifts in heroin ED50 measured by antinociceptive testing (Figure 4AꢀC). Although
vaccineꢀmediated shifts in heroin ED50 declined over the next three months, a second and third round of
vaccinations at months 5 and 8 maintained vaccine efficacy at approximately 50ꢀ70% of the initial level (Figure
4AꢀC). As a means to corroborate the antinociception results, ELISA titer was evaluated with the caveat that
sera binding to an immobilized drug hapten does not necessarily equate to affinity for the actual drug
molecule.40ꢀ42 To address this potential limitation, individual heroin ED50s were plotted against antiꢀheroin
titers, and a linear relationship was observed (Figure 4D). The correlation implies that the degree of serum
antibody binding to HerCOOH hapten is representative of the degree of binding to the actual opioids (heroin
and 6AM) in vivo.
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A possible liability in using TT in the final formulation is that preexisting immunity to TT from the
publically administered DTaP vaccine could reduce the subsequent response to a heroin vaccine, a phenomenon
known as carrierꢀinduced epitopic suppression (CIES);43ꢀ44 however, no evidence was found for the occurrence
of CIES in the context of the heroin vaccine (Figure S4).
Figure 4. Heroin conjugate vaccine shows robust, long-term efficacy in mice. (A) Heroin ED50 in the hot plate and tail immersion
tests at various time points following three rounds of vaccination (expressed as mean ± SEM). Mice (n = 6) were vaccinated i.p. with
50 µg HerCOOHꢀTT + 30 µg CpG 1826 at wk 0, 2 and 4; 25 µg HerCOOHꢀTT at wk 19, 21 and 23; 25 µg HerCOOHꢀTT + 15 µg
CpG 1826 at wk 31, 33 and 35. All immunizations contained 0.75 mg alum. #P < 0.05, ###P < 0.01, ###P < 0.001 (tail immersion); *P <
0.05, ***P < 0.001(hot plate) versus the 1 month time point. Foldꢀshifts in ED50 versus nonꢀvaccinated control mice are reported
above each set of data points. (B) Timeline of midpoint titers versus HerCOOHꢀBSA coating antigen over a period of 37 weeks.
Arrows indicate times of vaccination, while (T) indicates behavioral tests shown in the above panel. (C) Raw curves corresponding to
panel A. Heroin doseꢀeffect curve data expressed as mean ± SEM from six mice were generated via cumulative s.c. heroin dosing
(same procedures as Figure 3). (D) Correlation between heroin ED50s and midpoint titers. Pearson’s correlation: P < 0.0001 for both
tests.
Heroin vaccine-induced antibody response in non-human primates (NHPs)
As an initial assessment of the lead vaccine in NHPs, a pilot vaccine study was performed in which two
rhesus monkeys (M1,2) received the heroin conjugate while one monkey (M3) received the unmodified TT
carrier protein as a control. Following three injections, a significant and consistent antiꢀdrug IgG antibody
response was observed in the conjugateꢀvaccinated monkeys while antiꢀheroin antibodies were not observed in
the carrierꢀvaccinated monkey (Figure S5A). Preliminary behavioral assessments suggested that heroin doseꢀ
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effect curves were rightꢀshifted >3ꢀfold from baseline in both conjugateꢀvaccinated monkeys (Figure S5B).
Surface plasmon resonance (SPR) analysis of both monkey and mouse serum indicated subꢀmicromolar and low
nanomolar competitive IC50s for heroin and 6AM, respectively (Figure 5A), which in contrast to ELISA is
representative of actual antibody Kd.45 In comparing binding selectivity for other opioids, antiserum affinity for
morphine, oxycodone and methadone was >1000ꢀfold lower (Figure 5B).
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An extended vaccination study involving the same monkeys including one new one (M4) demonstrated that
all subjects produced a longꢀlasting, high IgG titer response to the vaccine (Figure 5C). Interestingly, the two
monkeys that received the heroin vaccine in the pilot study (M1,2) showed significantly higher titers than the
monkeys that received carrier (M3) or no vaccine (M4). SPR analysis of antiserum from each monkey revealed
consistent drug affinity for 6AM of ≤1 nM in monkeys M1,2 while monkeys M3,4 gradually increased 6AM
affinity over the course of the study to ~6 nM (Figure 5D). The 6AM affinity of M3,4 matches the 6AM affinity
of M1,2 observed after the pilot study (Figure 5A), while 6AM affinity in M1,2 increased further by
approximately sixꢀfold following the second immunization study (Figure 5D).
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Figure 5. Heroin conjugate vaccine elicits a robust anti-drug antibody response in monkeys with high affinity and selectivity
for 6AM. (A) Binding curves of vaccinated monkey and mouse antiserum for heroin and 6AM as determined by SPR in triplicate.
Points represents the mean binding to immobilized HerCOOHꢀBSA following serum incubation with 12 dilutions of heroin or 6AM
competitors. Binding values were normalized to serum binding without competitor drug. Listed IC50s ± SEM were derived from a
nonlinear fit of the binding curves. Serum was pooled from n = 2 monkeys (M1,2) and n = 6 mice collected after three initial
immunizations. (B) Binding selectivity of mouse and monkey antiserum for various opioids at 100 µM ([6AM] = 10 µM) by SPR in
triplicate. Minimum selectivity factors relative to 6AM are shown above each bar. (C) Antiꢀheroin IgG titers of rhesus monkeys (M1ꢀ
4) vaccinated at the indicated time points (arrows) with 400 µg HerCOOHꢀTT, 600 µg CpG ODN 2006 and 5 mg alum. Midpoint
titers were determined in duplicate by ELISA against a HerCOOHꢀBSA coating antigen. No antiꢀheroin IgM titers were detected at
any point. Monkeys M1 and M2 were previously vaccinated with the heroin vaccine in a pilot study while M3 received unconjugated
TT. M4 was not pretreated. (D) Individual monkey antiserum affinity for 6AM over time by SPR. Points represent mean IC50 ± SEM
determined from a 12 point 6AM dilution curve similar to panel A methods. Time points match up with the panel C timeline.
Vaccine-mediated alteration of heroin pharmacology in NHPs
Given the strong presence of 6AMꢀneutralizing antibodies in vaccinated rhesus monkeys, further
experiments were conducted to evaluate the capacity of the antibodies to alter the pharmacodynamics (PD) and
pharmacokinetics (PK) of heroin. Muꢀopioid agonists consistently produce doseꢀdependent decreases in operant
responding that appear to be mediated by pharmacologically similar populations of MOR. These MOR mediate
other muꢀopioid agonist behavioral effects, such as antinociceptive and discriminative stimulus effects.46ꢀ47
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Moreover, drug ED50 values can be quantified from operant responding to serve as an efficacy metric for MOR
modulators such as the antagonist NTX;48ꢀ50 therefore, an assay of scheduleꢀcontrolled responding (SCR) was
selected as a reliable behavioral measure of opioid pharmacology upon which to examine the
‘immunoantagonist’ vaccine.
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Following the doubleꢀdetermination of baseline heroin and oxycodone potencies to decrease rates of
responding (represented by ED50 values), monkeys were vaccinated and retested six weeks later, resulting in a
clear 3.5ꢀfold heroin ED50 shift (Figure 6A). A greater potency ratio was detected 2ꢀ3 weeks following week 11
(4.3ꢀfold) and 18 (4.1ꢀfold) booster injections (Figure 6A, S5C). The heroin ED50 values at these times (weeks
14 and 21) were similar to heroin ED50 values obtained following acute NTX pretreatment. Not only were
heroin potency ratios significantly increased versus baseline levels, they were also selectively elevated
compared to the control opioid, oxycodone, throughout the entire study (Figure 6A, S6). A correlation between
monkey antiꢀheroin titers and heroin SCR ED50 values revealed a linear relationship (Figure 6C). Notably, the
previously vaccinated monkeys M1 and M2 showed higher titers and ED50s compared to monkeys M3 and M4
in the SCR procedure (Figure S5C); however, normalization of all postꢀvaccination data to the preꢀvaccine
baseline revealed a relatively uniform antiꢀheroin behavioral effect among all four monkeys (Figure 6A).
To support the vaccineꢀmediated effects observed in the SCR assay, a heroin PK study was performed in the
four monkeys both before and after vaccination (Figure 6D). Results indicate that antiꢀdrug antibodies bound
large amounts of 6AM in the blood causing significant increases in PK metrics such as halfꢀlife and especially
AUC (19ꢀfold greater) compared to baseline measurements (Figure 6D,E); interestingly, when M1,2 were tested
after revaccination, a much larger AUC shift (193ꢀfold) was observed. The Cmax values were similarly shifted
by 13ꢀ and 167ꢀfold for the first and second vaccination studies, respectively. It should also be noted that no
gross undesirable effects to repeated vaccine administration were observed throughout the entire monkey study
(Figure S7).
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Figure 6. Heroin vaccine diminishes heroin potency and alters 6AM pharmacokinetics in rhesus monkeys. (A) Timeline of
changes in either heroin or oxycodone ED50s in monkeys after heroin vaccine administration. Monkeys (M1ꢀ4) were vaccinated i.m. at
the indicated time points (arrows) with 400 µg HerCOOHꢀTT, 600 µg CpG ODN 2006 and 5 mg alum. Baseline heroin and
oxycodone ED50 values were doubleꢀdetermined in each monkey prior to vaccination in the assay of scheduleꢀcontrolled responding
(SCR). Each point in the figure represents the average ratio of heroin (red) or oxy (blue) ED50 ± SEM relative to the baseline value.
Group mean ± SEM baseline ED50 values for heroin and oxycodone were 0.08 ± 0.03 and 0.19 ± 0.06 mg/kg, respectively. RM oneꢀ
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way ANOVA of heroin and oxycodone SCR over time F12,36 = 2.76 and 1.07, P = 0.0092 and 0.414, respectively; halfꢀfilled (P <
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0.05) and fullyꢀfilled (P < 0.01) circles indicate significance by Dunnett’s postꢀhoc test vs. heroin baseline. RM twoꢀway ANOVA of
heroin versus oxycodone SCR over time F1,3 = 13.2, P = 0.0358; halfꢀfilled (P < 0.05) and fullyꢀfilled (P < 0.01) squares indicate
significance by Bonferroni’s postꢀhoc test versus heroin. (B) SCR cumulative heroin doseꢀeffect curves at week 0 (baseline) and 14
(vaccine) in comparison to naltrexone (NTX) treatment at 3.2 µg/kg (1x) and 32 µg/kg (10x). Points represent mean ± SEM for n = 4
monkeys. (C) Correlation between titers against heroinꢀBSA and SCR heroin ED50 values collected over 26 weeks. P < 0.0001 by
Pearson’s correlation. (D) 6AM serum concentrations over time in n = 4 rhesus macaques (M1ꢀ4) following 0.32 mg/kg i.m. heroin.
The PK study was performed in the same subjects before and after a course of three initial immunizations. F1,6 = 20.35, P = 0.0041 by
a RM twoꢀway ANOVA comparing pre and post vaccination PK (postꢀvacc 1). After 7 months, monkeys M1 and 2 received three
additional immunizations and the PK study was repeated (postꢀvacc 2). (E) 6AM AUC values corresponding to the PK study. Fold
increases versus baseline are reported. **P < 0.01 by paired tꢀtest. Cmax values (ng/mL): baseline = 32.5 ± 6.8, postꢀvacc 1 = 418 ±
110, postꢀvacc 2 = 5430 ± 2800.
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Discussion
Achievement of heroin vaccine effectiveness required mastery of a number of unique challenges. First, the
desired immune response from the heroin vaccine is distinct compared to vaccines being developed against
pathogens because drug vaccines have a greater requirement for strong humoral immunity; a larger molar
quantity of drug relative to pathogens must be neutralized by IgGs for the drug vaccine to achieve efficacy. On
the other hand, while cellꢀmediated immunity is necessary for subduing diseaseꢀcausing pathogens, it would be
counterproductive in the development of immunoantagonists for the treatment of substance use disorders.
Second, utilizing TT as a carrier protein at the clinical level could readily be questioned if preꢀexisting
immunity to TT diminished the immune response to the drugꢀcarrier conjugate. Fortunately, preꢀexisting
antibody titers to TT did not suppress heroin vaccine efficacy. Third, as heroin is a prodrug for 6AM/morphine,
hapten design was tailored to direct antibody binding toward 6AM followed by heroin but not morphine.
Despite being a psychoactive metabolite of heroin, morphine penetrates the bloodꢀbrain barrier much less
readily, hence, antibody sequestration of heroin/6AM until enzymatic hydrolysis ensues is key to vaccine
performance.51 Fourth, while titer is typically the metric used in gauging vaccine success, antibody affinity is
also critical. The low nanomolar antibody affinity for 6AM, as observed by SPR, provides a competitive sink
with the brain MOR for drug binding based on Le Chatelier’s principle, causing heroin potency reduction in
behavioral assays.
Heroin hapten design was greatly improved through conversion of the terminal thiol (HerSH) to a
carboxylic acid (HerCOOH). This modification enabled a more efficient and reliable protein coupling,
producing a more efficacious immunoconjugate with higher epitope density. Furthermore, the HerCOOH
hapten contains a shorter, more ‘immunologically silent’ linker with less chemical functionalities relative to
HerSH and HerdBA. Following antigen processing, the HerCOOH hapten linker likely interferes minimally
with immune presentation of a heroinꢀlike epitope. Previous studies have shown that peptidic linkers can
increase antiꢀhapten immune responses,52ꢀ54 possibly by enhancing hapten anchoring to the MHCII55 (Figure 2).
However, the dBA linker was not effective, which may be explained by the fact that betaꢀalanine is not
naturally found in proteins, thus hampering immune processing and presentation of HerdBA. Because it was not
clear whether heroin or 6AM should be emulated for hapten design, as heroin and 6AM are equipotent,4ꢀ6
a
direct comparison was made between the corresponding haptens (HerCOOH and 6AMCOOH). From an
efficacy standpoint, both 6AMCOOHꢀ and HerCOOHꢀTT conjugates behaved similarly, implying that the
heroin hapten likely hydrolyzes in vivo almost completely to the 6AM hapten. This result is not unexpected
given the known high lability of the 3ꢀacetyl group and is further supported by the fact that antibodies show
drug affinity in the following order: 6AM>heroin>>morphine. Moreover, the probable 3ꢀacetyl hydrolysis of
our hapten corroborates the dynamic nature of the vaccine as we had previously posited,36ꢀ37 although the 6ꢀ
acetyl group likely remains mostly intact given the low antiserum affinity for morphine. Finally, while several
heroin vaccines containing hydrolytically stable haptens are currently in preclinical development, they have yet
to demonstrate blockade of heroin effects beyond a 1 mg/kg dose in rodents and have not been tested in
NHPs.51, 56ꢀ59
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In exploring dosing and adjuvants for the HerCOOHꢀTT conjugate, a positive correlation was discovered
between vaccine efficacy and the amount of immunoconjugate and adjuvant CpG ODN. Although a plateau
may exist in the doseꢀresponsive effect of the immunoconjugate and CpG ODN, no such plateau was observed
at the doses tested in mice. The implication of this finding is that the vaccine dosage can be increased to achieve
a higher degree of efficacy. In fact, conjugate doses as high as 2000 ꢁg were safely used in a clinical cocaine
vaccine study.34 By comparison, a relatively low conjugate dose (330ꢀ400 µg) was used in our NHP studies. In
terms of the primateꢀcompatible TLR9ꢀagonist (CpG ODN 2006), a dose of 400ꢀ600 µg was used in our studies
similar to a nicotine vaccine study,60 while doses of up to 2 mg have been safely used.61 Promisingly, CpG
ODN 2006 has shown success in human vaccine trials (phase I and II) with a wide variety of antigens.62ꢀ66 In
contrast, we found that cyclic dinucleotide cGAMP only presented mild activity as a vaccine adjuvant at the
doses tested, despite sharing structural and mechanistic similarities with CpG ODN – both are DNAꢀbased and
act as pathogenꢀassociated molecular patterns (PAMPs) to stimulate the innate immune system. Likely, the
strong activation of Bꢀcells via TLR9 to produce an antiꢀdrug humoral response contributes to the success of
CpG ODN 1826 and 2006 as adjuvants.67ꢀ68 These effects were synergistic with alumꢀmediated induction of
humoral immunity, as shown previously.35, 60, 69ꢀ70 As only one dosage of CpG ODN and alum was investigated
in the current study, fine tuning of CpG ODN and alum dosing in future monkeys studies would be anticipated
to further enhance vaccine efficacy.
Once a heroin vaccine formulation was identified that was efficacious in generating longꢀterm, antibodyꢀ
mediated protection from heroin PD, studies in rhesus macaques were pursued to evaluate the clinical potential
of the vaccine. One major hurdle for drug vaccines in human trials is achieving efficacy in all patients.
Although clinical trials for nicotine and cocaine vaccines have failed to demonstrate significant efficacy, they
did demonstrate a proofꢀofꢀconcept that patients who generated a strong antiꢀdrug antibody response displayed
protection from the abuseꢀrelated drug effects.28ꢀ31 In order to improve drug vaccine performance, preclinical
testing must employ clinically relevant benchmarks focusing directly on vaccine capacity to selectively alter the
behavioral pharmacology of the target drug. Our studies in an outbred strain of mice (Swiss Webster)
demonstrated that the vaccine reliably induced antiꢀdrug titers and significantly shifted the heroin doseꢀeffect
curves in the antinociception assay. In fact, the only detectable source of immune variability was the degree of
hapten conjugation; heroin conjugates with greater haptenation produced greater efficacy upon immunization,
which has been observed previously.56, 71
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The optimized vaccine formulation identified from the mouse studies translated well to rhesus monkeys,
albeit with smaller heroin potency shifts, possibly due to immunological differences between species or vaccine
dosages. Moreover, the large initial spike in vaccine response that was only observed in mice can be explained
by the differing vaccine injection routes, i.p. in mice vs. i.m. in monkeys, and in the former case, the vaccine
can rapidly drain to the spleen to induce a strong, shortꢀlived Bꢀcell response. Regardless, significant antiꢀheroin
IgG titers with low nanomolar 6AM affinity were observed in all four monkeys. In addition, the humoral
immune response to the vaccine significantly altered heroin PK, increasing 6AM AUC values by ~19ꢀfold and
halfꢀlife by ~3ꢀfold, resulting in the observed reduction in heroin PD. The PK results are in agreement with
previous studies which have noted that conjugateꢀvaccinated animals showed higher drug concentrations in
serum due to antibody sequestration leading to lower drug concentrations in the brain relative to controls; a
phenomenon observed for opioids45, 52, 72ꢀ73 and other drugs such as methamphetamine, cocaine and nicotine.71,
74ꢀ77
In a manner that has not been fully studied, the drugꢀspecific antibodies appear to increase drug halfꢀlife
and diminish drug clearance. This is likely caused by antibodyꢀdrug binding, which hampers glomerular
filtration of drugs by the kidneys and shields drugs from metabolic destruction. Eventual drug metabolism and
clearance does not appear to be mediated by immunological mechanisms but rather by slow dissociation of
antibodyꢀdrug complexes.72
As a testament to vaccine durability, the two monkeys M1,2 that participated in a second study ~7 months
later showed much greater antibody titer, affinity and 6AM AUC, indicating that vaccination in the second
study strongly recalled a memory immune response initially established during the first study. In considering
dosing schedule, the data suggests that at least three vaccinations spaced 6 weeks apart or greater is required for
achieving significant immunity against heroin; however, as observed in monkeys M1,2, vaccination on an even
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longer time scale may be optimal. Importantly, no evidence of immune tolerance to the vaccine was seen at any
point despite multiple immunization rounds and frequent administration of heroin as a result of SCR testing.
The immunochemical and PK data support the heroin SCR data, the latter of which revealed an approximately
4ꢀfold shift in the heroin doseꢀeffect curve following each immunization. While the decrease in heroin potency
was observed via the i.m. route of administration, these results are unlikely to be significantly different than the
i.v. route commonly used by humans. The relative ratios of heroin, 6AM, and morphine after i.m.
administration were consistent with the PK profile observed following i.v. heroin administration in humans.78
Furthermore, our first generation vaccine was effective in attenuating i.v. heroin selfꢀadministration in
rats.37 Specifically, the vaccine could block heroin reinstatement following a single bolus of 0.18 mg/kg i.v.
heroin, which would translate to a 14.4 mg dose in an 80 kg adult. This dose is within the dose range reported
by heroin users (see Erowid.org). As our second generation vaccine has been shown to be at least threeꢀfold
better than the first generation vaccine in the mouse antinociception model, we posit the second generation
vaccine's capacity to neutralize i.v. heroin would only be enhanced. Consolidation of our data strongly suggests
that the vaccine acts as a heroin immunoantagonist; furthermore, the vaccine effects paralleled the effects of the
FDAꢀapproved opioid antagonist NTX. In previous rhesus monkey studies, NTX antagonized the rate
suppressant, antinociceptive and discriminative stimulus effects of heroin.46, 79ꢀ80 Similarly, reductions in heroin
vs. food choice have been demonstrated by administration of another opioid antagonist naloxone in nonꢀopioid
dependent monkeys81 and NTX pretreatment in nonꢀopioid dependent rats.82 These results provide empirical
evidence that the vaccineꢀmediated antagonism of heroin in the SCR assay would be predictive of antagonism
of heroin effects in other models such as drug discrimination and selfꢀadministration. Given the noteworthy
vaccine results in the SCR procedure, future studies to determine vaccine effects in more complex NHP
behavioral procedures, e.g. i.v. selfꢀadministration, are warranted.
Other studies in NHPs have demonstrated the efficacy of optimized nicotine and cocaine vaccines. The
second generation nicotine conjugate (NIC7ꢀCRM) was optimized in mice83 and translated well to cynomolgus
monkeys in a CpG ODN + alum formulation to elicit sustained (~106) antiꢀnicotine titers.84 Following a nicotine
challenge in vaccinated monkeys, blood nicotine Cmax and AUC increased by 29 and 89ꢀfold, respectively,84 and
nicotine brain concentrations were reduced.71 A fully synthetic nicotine vaccine (SELꢀ068) also translated from
mice to monkeys85 and has been shown to attenuate nicotine discrimination in squirrel monkeys while reducing
nicotine potency by 3ꢀfold.86 The cocaine hapten, which we’ve termed GNE conjugated to a disrupted
adenovirus (dAd5) and formulated with a proprietary adjuvant (Adjuplex) has consistently generated high antiꢀ
cocaine titers (105ꢀ106) in a number of rhesus macaque studies.87ꢀ90 This vaccine increased cocaine Cmax in
serum by ~3.5ꢀfold, mitigated biodistribution of cocaine to the brain and other organs,88ꢀ89 and attenuated
cocaine selfꢀadministration and reacquisition.90 Although these reports provide some context for our NHP
heroin vaccine studies, direct comparisons between our studies and others are limited. In all cases, antiꢀhapten
titer levels showed similar behavior and magnitude in response to the vaccines; however, our studies use
midpoint titers, which are 10ꢀ100 fold lower than the endpoint titers used in other studies. Furthermore, haptenꢀ
specific titer levels do not necessarily reflect affinity to the actual target drug, and thus are not always indicative
of efficacy, although a robust correlation between titer and efficacy was observed for the disclosed heroin
vaccine. While previous NHP vaccine studies have not fully investigated drug affinity of monkey antiserum, we
have observed 0.5ꢀ6 nM affinity to 6AM. Comparatively, dAd5GNE in NHPs was shown to produce 5ꢀ120 nM
antibody affinity to cocaine.87 In considering behavioral testing, few studies have compared drug ED50s in
vaccinated and nonꢀvaccinated animals while we have demonstrated heroin doseꢀeffect curve shifting in both
mouse and monkey models. For reference, the SELꢀ068 vaccine reduced nicotine potency by 3ꢀfold in monkeys
while we saw a 4ꢀfold reduction in heroin potency. However, results in the more complex selfꢀadministration
procedures in a cocaine vaccine study90 and the first heroin vaccine study,24 cannot be related to the ED50
determinations in our study. On the other hand, PK metrics in vaccinated monkeys can be compared with the
caveat that drug PK/PD between each of the drugs differ drastically. Vaccineꢀmediated foldꢀincreases in heroin
Cmax for HerCOOHꢀTT (postꢀvacc 1) appeared to be half that of the nicotine Cmax for NIC7ꢀCRM84 but fourꢀ
fold greater than cocaine Cmax for dAd5GNE;89 therefore, our vaccine possesses excellent serum neutralizing
capacity for heroin and its psychoactive metabolites relative to other vaccines.
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In response to the opioid epidemic that has plagued the United States, the disclosed heroin vaccine could
satisfy a dire, unmet need for an opioid use disorder therapeutic. Since the vaccine reduces heroin potency,
vaccinated drug users would encounter an increased cost to “getting high” potentially similar to clinically
available and FDAꢀapproved depot NTX formulations.91ꢀ92 Indeed, our previous studies have shown that
following a period of drug abstinence, drugꢀdependent, immunized rats actually extinguish selfꢀadministration
of heroin,37 and these rats demonstrated 4ꢀfold shifted heroin doseꢀeffect curves in antinociceptive testing.
Considering this study and the similar curve shifts observed in monkeys, our vaccine has the potential to be
clinically useful; it may serve as a ‘safety catch’ for preventing relapse episodes in former heroin users
attempting to maintain drug abstinence, or it may help current heroin abusers to achieve abstinence. This
hypothesis is supported by clinical studies showing that longꢀacting depot NTX mitigated the reinforcing effects
of heroin in humans,92ꢀ93 and instead of heroin users increasing their drug intake to surmount the antagonist,
heroin extinctionꢀlike behaviors occurred.91, 94 The obvious major benefits to vaccination over pharmacological
antagonists are the potential for increased duration of action and decreased side effects. Coꢀadministration of
opioid agonists methadone or buprenorphine as needed along with the vaccine would likely enhance therapeutic
efficacy by alleviating opioid cravings. Such combination therapy would be possible due to the vaccine’s
selective sequestration of 6AM by >1000ꢀfold over other opioids; moreover, prescription pain medication e.g.
oxycodone also would not interact with the heroin vaccine.
In conclusion, an efficacious heroin vaccine has been identified through optimization of the adjuvant (CpG
ODN + alum), carrier protein (TT) and hapten (HerCOOH). The vaccine is efficacious in basic preꢀclinical
mouse and NHP models over a wide range of heroin doses, accomplishing an important milestone in the drug
development process to human clinical trials. Forty years after the first report of a heroin/morphine vaccine,24
numerous studies and research groups have advanced the concept of antiꢀdrug vaccines to a functional level,
whereby the vaccines act as a longꢀterm ‘immunoantagonist’ to attenuate drug PD. While we have speculated as
to how our particular heroin vaccine could be used to treat use disorder, future studies involving more advanced
NHP models and clinical trials must be performed to elucidate the true therapeutic utility of this vaccine as well
as other opioid vaccines.
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Associated Content
Supporting Information
Materials and methods, conjugate mass data, titer data, additional mouse and monkey vaccine data: Tables S1ꢀ2, Figures S1ꢀ10. This
information is available free of charge on the ACS Publications website at http://pubs.acs.org.
Author Information
Corresponding Authors
*kdjanda@scripps.edu
Notes
The authors declare no conflicts of interest.
Acknowledgments
This work was supported by the NIH under grants UH2DA041146, R01DA026625, K99DA037344 and F31DA037709 and the
Skaggs Institute for Chemical Biology. This is manuscript # 29467 from The Scripps Research Institute.
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