International Immunopharmacology 16 (2013) 50–56
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Enhancing immunogenicity of a 3′aminomethylnicotine-DT-conjugate anti-nicotine vaccine with CpG adjuvant in mice and non-human primates Michael J. McCluskie a,⁎, David C. Pryde b, David P. Gervais c, 1, David R. Stead c, Ningli Zhang a, Michelle Benoit a, Karen Robertson a, In-Jeong Kim a, Tharsika Tharmanathan a, James R. Merson c, Heather L. Davis a a b c
Pﬁzer Vaccine Research, Ottawa, ON, Canada Pﬁzer Worldwide Medicinal Chemistry, Cambridge, UK Pﬁzer Vaccine Research, La Jolla, CA, USA
a r t i c l e
i n f o
Article history: Received 6 February 2013 Received in revised form 20 March 2013 Accepted 20 March 2013 Available online 2 April 2013 Keywords: Nicotine Vaccine CpG Immunotherapy Adjuvant Immunization
a b s t r a c t Tobacco smoking is one of the most preventable causes of morbidity and mortality, but current smoking cessation treatments have relatively poor long term efﬁcacy. Anti-nicotine vaccines offer a novel mechanism of action whereby anti-nicotine antibodies (Ab) in circulation prevent nicotine from entering the brain, thus avoiding the reward mechanisms that underpin nicotine addiction. Since antibody responses are typically long lasting, such vaccines could potentially lead to better long-term smoking cessation outcomes. Clinical trials of anti-nicotine vaccines to date have not succeeded, although there was evidence that very high anti-nicotine Ab titers could lead to improved smoking cessation outcomes, suggesting that achieving higher titers in more subjects might result in better efﬁcacy overall. In this study, we evaluated CpG (TLR9 agonist) and aluminum hydroxide (Al(OH)3) adjuvants with a model anti-nicotine antigen comprising trans-3′ aminomethylnicotine (3′AmNic) conjugated to diphtheria toxoid (DT). Anti-nicotine Ab titers were signiﬁcantly higher in both mice and non-human primates (NHP) when 3′AmNic-DT was administered with CpG/Al(OH)3 than with Al(OH)3 alone, and afﬁnity was enhanced in mice. CpG also improved functional responses, as measured by nicotine brain levels in mice after intravenous administration of radiolabeled nicotine (30% versus 3% without CpG), or by nicotine binding capacity of NHP antisera (15-fold higher with CpG). Further improvement should focus on maximizing Ab function, which takes into account both titer and avidity, and this may require improved conjugate design in addition to adjuvants. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Tobacco use remains the world's leading cause of preventable death, killing nearly six million people annually . Current pharmacotherapies typically either replace the source of nicotine (nicotine replacement therapy such as gums, patches, etc.) or act on sites in the central nervous systems to reduce nicotine reward and/or withdrawal symptoms. Treatment periods are relatively brief (typically up to 12 weeks) and relapse is prevalent, especially after treatment ends, such that long-term outcomes remain poor, with only 12–22% of treated subjects remaining abstinent at the end of one year [2,3]. Anti-nicotine vaccines induce nicotine-speciﬁc antibodies that bind nicotine in the periphery and prevent its entry to the brain,
⁎ Corresponding author at: Pﬁzer Vaccine Research, Ottawa Laboratories, 340, Terry Fox Drive, Suite 200, Ottawa, ON K2K 3A2, Canada. Tel.: +1 613 254 5207; fax: +1 613 254 5625. E-mail address: [email protected]
ﬁzer.com (M.J. McCluskie). 1 Present address: Health Protection Agency, Process and Analytical Development Group, Porton Down, Salisbury, UK. 1567-5769/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.intimp.2013.03.021
thereby preventing the interaction of nicotine with its receptor. If sufﬁcient nicotine is kept from the brain, it is expected that the pharmacological induction of the reward sensation would be prevented and help break the addiction cycle associated with smoking. Since antibodies induced in response to vaccines are of long durations (typically months to years), this approach may also result in lower relapse rates over time . A hapten, such as nicotine, is a small molecule that can elicit an immune response only when attached to a large carrier such as a protein that is required to provide T-help and a scaffold for antigen presentation. Once anti-hapten responses are induced, the small-molecule hapten (i.e., nicotine) can bind to the antibody, but it will not usually boost the immune response. Clinical trials have been conducted with several different anti-nicotine vaccines that utilize nicotine-derived haptens conjugated to different carriers, and most use the same adjuvant (Al(OH)3). Two vaccines underwent phase 2 clinical testing, and both Nic-Qb  and NicVax  failed to show efﬁcacy in the intent to treat (ITT) population for long-term continuous abstinence rate (CAR) at 1 year, counting from the ﬁrst dose. However, in both studies, subgroup analyses provided proof of mechanism, in that the top 30% of responders for area under
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the curve of antibody titers from the time of the ﬁrst dose until 26 weeks for NicVax, or from 3 months to 6 months for NicQb  had signiﬁcantly enhanced 52-week CAR compared to placebo, with an odds ratio of approximately 2 in both studies [5,6]. These results suggested that vaccine efﬁcacy might be achieved in the ITT population if high antibody levels could be generated in a greater proportion of subjects. A number of different approaches have been tested in animal models and shown to improve immunogenicity. These include modiﬁcations of hapten structure, linker composition and position, choice of carrier, route of administration and co-administration of more than one nicotine hapten conjugate [7–13]. Another approach may be through the use of adjuvants. Adjuvants are commonly added to antigens to augment antibody titers. Aluminum salts are widely used as adjuvants to enhance humoral immunity to vaccines, and Al(OH)3 was the adjuvant used with both NicVax and NicQb. Numerous studies with multiple antigens have shown that combinations of adjuvants are usually more effective than single adjuvants, especially when a delivery system such as an aluminum salt is combined with an immune modulator such as an agonist for a Toll-like receptor (TLR). The combination of Al(OH)3 and a CpG oligodeoxynucleotide (CpG) that activates via TLR9 can enhance antibody titers 10-fold or more over either adjuvant alone in mice . In humans, CpG combined with Al(OH)3 enhanced antibody titers against four different antigens by at least 5-fold compared to Al(OH)3 as sole adjuvant,[15–18] and, in one study where it was evaluated, also signiﬁcantly enhanced antibody afﬁnity. Herein, we describe our ﬁndings in mice and monkeys testing the ability of CpG to enhance anti-nicotine antibody functionality induced in response to a model anti-nicotine vaccine comprised of trans-3′ aminomethylnicotine conjugated to diphtheria toxoid (3′AmNic-DT) as antigen and Al(OH)3 as adjuvant.
(Ag) in combination with Al(OH)3 (40 μg Al 3 +) alone or together with CpG (50 μg) made up to a total volume of 50 μL with phosphate buffered saline (PBS; Sigma Chemical Co., St. Louis, MO). CpG alone was not tested as adjuvant since pilot studies had shown that CpG gave weaker responses alone than in combination with Al(OH)3, as has been seen with other antigens . Preliminary studies had also determined that these doses of antigen and adjuvant were suitable for use in mice. The vaccine formulations were administered by intramuscular (IM) injection to the left tibialis anterior (TA) muscle of mice lightly anesthetized with Isoﬂurane® (CDMV, St. Hyacinthe, QC) on days 0, 28 and 42. Animals were bled on days 28, 42 and 54 by submandibular venous puncture using heparin as an anti-coagulant and the recovered plasma was used for quantitation of nicotine-speciﬁc immune responses. Longevity of anti-nicotine antibody responses was determined in a separate set of animals which were immunized in an identical manner but were bled at additional time-points up to week 20 post ﬁrst immunization. All mouse experiments were repeated on at least one independent occasion to ensure reproducibility of results. Cynomolgus monkeys (LAB Research Inc., Montreal, QC, n = 5/ group) were immunized on days 0, 28, 56, 189 and 280 with 100 μg 3′ AmNic-DT, in combination with Al(OH)3 (400 μg Al3+) as sole adjuvant or combined with CpG (500 μg). All vaccine formulations were made up to a total volume of 0.6 mL with PBS and administered by intramuscular (IM) injection in the quadriceps muscle. At multiple time-points up to 44 weeks post-prime, animals were bled by saphenous venous puncture using sodium citrate as an anti-coagulant and recovered plasma was used for quantitation of nicotine- and DT-speciﬁc antibodies. All animal studies were conducted in AAALAC accredited facilities and were subject to approval by the local Institutional Animal Care and Use Committees. 2.4. Anti-nicotine antibody ELISA
2. Materials and methods 2.1. Antigen Diphtheria toxoid (DT; Pﬁzer, Lincoln, NE) in Dulbecco's Phosphate Buffered Saline (DPBS) was ﬁrst derivatized with succinic anhydride (Acros, Pittsburgh, PA) added as powder. The reaction mixture was incubated for 2 h at room temperature and post-incubation un-reacted succinic anhydride and reaction by-products were removed by buffer exchange into DPBS (using a Nap-25 Column, Pierce, Rockford, IL). An excess of Trans-3′aminomethylnicotine (3′AmNic, Toronto Research Chemicals, North York ON), solubilized in DPBS was added to the succinylated DT, along with an equal weight of 1-ethyl-3-(3diethylamino)propyl carbodiimide hydrochloride (EDC) (Pierce, Rockford, IL) and sulfo N-hydroxysuccinimide (sNHS) (Sigma Aldrich, St Louis, MO). The conjugation reaction was incubated for 2 h at room temperature to yield the 3′AmNic-DT conjugate. Post-conjugation, unreacted reagents and reaction bi-products were removed by buffer exchange into DPBS (using a Nap-25 Column, Pierce) prior to storage at 2–8 °C. 2.2. Adjuvants The B Class CpG ODN (CpG) of sequence 5′ TCG TCG TTT TTC GGT GCT TTT 3′ was synthesized with a nuclease-resistant phosphorothioate backbone (Pﬁzer, St. Louis, MA) . Aluminum hydroxide (Al(OH)3) was used in the form Alhydrogel “85”, which was obtained from Brenntag Biosector (Denmark).
The levels of anti-nicotine antibodies in mouse or monkey plasma were quantiﬁed using an ELISA assay as follows: A nicotine derivative (rac-trans3′-thio methyl nicotine dihydrochloride, Toronto Research Chemicals) was conjugated to bovine serum albumin (BSA, SigmaAldrich) to obtain nicotine-BSA and used to coat 96 well Immuno Maxi-Sorp ELISA plates (VWR) (100 μL/well) at a ﬁnal concentration of 1 μg/mL in carbonate–bicarbonate buffer (Sigma Aldrich). Plates were incubated overnight at 4 °C, aspirated and washed with PBS containing 0.05% Tween 20 (Sigma-Aldrich). Plates were blocked with 200 μL of blocking buffer (carbonate–bicarbonate buffer + 10% Bovine Calf Serum, BCS, Fisher) at 37 °C for 1 h and then washed as above. Samples were serially diluted in dilution buffer (1× PBS with 0.05% Tween 20 + 10% BCS) and added to the plates (100 μL/well). Plates were incubated at 37 °C for 2 h. The plates were washed again and then incubated with Goat Anti-mouse IgG-HRP or Mouse Anti-human IgG-HRP, for mouse and monkey samples, respectively, (Southern Biotech, New Orleans, LA), diluted with dilution buffer (1× PBS with 0.05% Tween 20 + 10% BCS) for 1 h at 37 °C. The plates were then washed again and incubated with O-phenylenediamine dihydrochloride (OPD) substrate (1 × 5 mg OPD tablet dissolved in Phosphate Citrate Buffer, Sigma-Aldrich) in dark for 30 min at room temperature. The reaction was stopped by addition of 50 μL of 4 N Sulfuric Acid (VWR) to each well and read at 450 nm using automated plate reader. Titers were deﬁned as the highest plasma dilution that resulted in an absorbance value (OD 450) two times greater than that of non-immune plasma, with a cut-off value of 0.05. 2.5. Antibody afﬁnity by ammonium thiocyanate assay
2.3. Animal models and immunization Female BALB/c mice (Charles River Laboratories, Montreal, QC, n = 10/group) were immunized with 10 μg 3′AmNic-DT antigen
Afﬁnity of anti-nicotine antibodies was measured by an ELISA elution assay using ammonium thiocyanate (NH4SCN) as a chaotropic agent. ELISA plates were coated with nicotine-BSA conjugate, incubated
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overnight, washed and blocked as above. Next, plasma samples, previously determined to contain anti-nicotine antibodies, were diluted in PBS containing 0.05% Tween 20 + 10% bovine calf serum (BCS) to achieve optimal absorbance values of approximately 1.0 at 450 nm, and were then added to plates at 100 μL/well. The plates were incubated for 2 h at 37 °C and then washed. Next, elution was performed by adding NH4SCN (100 μL/well) in concentrations ranging from 0 to 2.0 M diluted in PBS/0.05% Tween 20 and incubated for 15 min at room temperature. The plates were then washed and antibody binding was detected as above. The percent reduction in binding of antigen–antibody (% reduction in OD) in the presence of NH4SCN was determined and plotted against the molar concentration of NH4SCN. Avidity index was calculated as the molar concentration of NH4SCN required to produce 50% reduction in binding.
containing 3 μCi 3H-nicotine in 100 μL of PBS. The 0.05 mg/kg dose of nicotine used is considered approximately equivalent to the mg/kg dose of nicotine delivered to a human by three smoked cigarettes . Blood was obtained 5 min later via cardiac puncture and plasma collected. The mouse was immediately perfused with PBS by injecting 20 mL into the left ventricle of the heart over 1–2 min and the brain harvested and weighed. The brain was digested at ~50 °C for 72 h in 1 mL digestion buffer (100 mM sodium chloride, 25 mM Tris, 25 mM EDTA, 0.5% Igepal CA-630 and 0.3 mg/mL proteinase K) per 100 mg tissue. Aliquots (100 μL) of brain digest or plasma were mixed with 5 mL liquid scintillation ﬂuid and levels of radiolabeled nicotine were determined by liquid scintillation counting. 2.8. Ex vivo functional assay: nicotine-binding capacity of anti-nicotine antibodies
2.6. Antibody avidity by competition ELISA For NHP samples, since a higher plasma volume was available, avidity was also determined using a competition ELISA method. ELISA plates were coated with nicotine-BSA conjugate, incubated overnight, washed and blocked as above. Thereafter, plasma samples, previously determined to contain anti-nicotine antibodies, were diluted in PBS containing 0.05% Tween 20 + 10% BCS to achieve absorbance values of approximately 1.0–1.5 at 450 nm; and nicotine (Sigma-Aldrich) was serially diluted starting at 20,000 μM. Equal volumes (65 μL) of diluted samples and nicotine were added to wells of a non-coated 96 well plate and allowed to incubate for 1 h at 37 °C. Following incubation, the plasma/nicotine mixtures were added at 100 μL/well to the previously blocked nicotine-BSA coated plates. The plates were incubated for 30 min at 37 °C and then washed again. Thereafter, detection of antibody binding proceeded as outlined above for anti-nicotine antibody ELISA. OD readings at 450 nm were plotted against the molar concentration of nicotine and the 50% inhibition (IC50) was extrapolated for each sample tested. 2.7. In vivo functional assay: nicotine distribution in brain and plasma The function of anti-nicotine Ab in immunized mice was evaluated by assessing nicotine distribution in the brain and plasma following tail vein infusion (b 5 s) of 0.05 mg/kg of nicotine hydrogen tartrate
2.9. Statistical analysis Data were analyzed using GraphPad Prism (GraphPad Software, San Diego, CA). Statistical signiﬁcance of the difference between two groups was calculated by Student's 2-tailed t-test on log-transformed data and between three or more groups by 1-factor analysis of variance (ANOVA) followed by post-hoc analysis. Differences were considered to be not signiﬁcant with p > 0.05.
Anti-NIC IgG (GMT+95%CI)
The function of anti-nicotine Ab in immunized NHP was evaluated by assessing nicotine-binding capacity using a modiﬁcation of a previously published method . In brief, plasma collected at various times during and after vaccine dosing was spiked with a ﬁxed amount of nicotine equivalent to high blood levels in smokers (100 ng/mL) and then subjected to equilibrium dialysis against DPBS for 3 h at 37 °C using Spectrapor 4 membranes with a molecular weight cutoff of 12 to 14 kDa and 20 Micro-cell Equilibrium Dialyzer (Spectrum Labs, Rancho Dominguez, CA). The 100 ng/mL spiking dose of nicotine was selected as this represents a high concentration of nicotine in the arterial blood of a heavy smoker. Using nicotine-d3 as internal standard, aliquots from the sample and buffer sides of dialysis membrane were extracted using a protein precipitation extraction procedure. The extracted samples were then injected into an HPLC equipped with Applied Biosystems API 4000 LC/MS/MS system and concentrations of bound and unbound nicotine determined.
Post 2nd dose
Post 3rd dose
Post 1st dose Post 2nd dose Post 3rd dose
Fig. 1. Anti-nicotine antibody titer and avidity in mice. BALB/c mice (n = 10/gp) were immunized by IM injection with 10 μg of 3′AmNic-DT adjuvanted with Al(OH)3 (40 μg Al3+) ± CpG (50 μg) on days 0, 28 and 42. Plasma was collected on days 28, 42 and 54 and anti-nicotine antibody levels determined by ELISA (Panel A) and antibody avidity by elution ELISA (Panel B). ⁎, ⁎⁎, ⁎⁎⁎ and ⁎⁎⁎⁎ represent p b 0.05, 0.01, 0.001 and 0.0001, respectively.
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Anti-nicotine IgG Titer
(p b 0.0001), indicating peripheral sequestration of the nicotine, as predicted by the purported mechanism of action. The proportion of nicotine kept from the brain was approximately 30%. Mice immunized with 3′AmNic-DT/Al(OH)3/CpG also had signiﬁcantly higher nicotine in the blood and lower nicotine in the brain than animals immunized with 3′AmNic-DT/Al(OH)3, in which only about 3% of nicotine was kept from the brain (Fig. 3A, B, p b 0.001). While there were trends for increased blood levels and reduced brain levels of nicotine in animals immunized with 3′AmNic-DT/ Al(OH)3 compared to non-immunized controls, these differences were not signiﬁcant (Fig. 3A, B; p > 0.05).
DT-Hapten + Al(OH) 3 DT-Hapten + Al(OH) 3/CpG 103 0
wks Fig. 2. Kinetics of anti-nicotine antibody response in mice. BALB/c mice (n = 10/gp) were immunized by IM injection with 10 μg of 3′AmNic-DT adjuvanted with Al(OH)3 (40 μg Al3+) ± CpG (50 μg) on days 0, 28 and 42. Plasma was collected at various time-points post immunization and anti-nicotine antibody levels determined by ELISA. ⁎, ⁎⁎, ⁎⁎⁎ and ⁎⁎⁎⁎ represent p b 0.05, 0.01, 0.001 and 0.0001, respectively.
3. Results 3.1. Effect of adjuvants on immunogenicity of 3′AmNic-DT in mice In mice, the addition of CpG to the 3′AmNic-DT/Al(OH)3 vaccine formulation signiﬁcantly increased nicotine-speciﬁc IgG levels (about 20-fold) following prime or boost doses (p b 0.05) (Fig. 1A). In addition, antibody afﬁnity was shown to be signiﬁcantly increased by CpG (p b 0.05) (Fig. 1B), and the ratio of IgG2a:IgG1 antibody isotypes was increased (0.4906 ± 0.2766 versus 0.0002 ± 0.0002, for CpG/Al(OH)3 and Al(OH)3, respectively, p b 0.01), which in mice is indicative of a more Th1-biased response (data not shown). Signiﬁcantly higher titers of nicotine-speciﬁc antibodies with CpG were maintained for up to ﬁve months post immunization, as shown in the separate 20-week study (Fig. 2). The in vivo function of the anti-nicotine antibodies in mice, as assessed by IV administration of radiolabeled nicotine, showed that addition of CpG resulted in signiﬁcantly improved results. The 3′ AmNic-DT/Al(OH)3/CpG formulation signiﬁcantly increased levels of nicotine in the blood (Fig. 3A) and signiﬁcantly decreased levels of nicotine in the brain (Fig. 3B) compared to non-immunized controls
Plasma nicotine (ng-eq/mL)
3.2. Effect of adjuvants on immunogenicity of 3′AmNic-DT in NHPs Cynomolgus monkeys immunized with 3′AmNic-DT/Al(OH)3/CpG had signiﬁcantly higher levels of anti-nicotine antibodies at all time-points following the ﬁrst boost than those immunized with vaccine without CpG (p b 0.05) (Fig. 4A). The afﬁnity of NHP anti-nicotine antibodies, with and without CpG in the vaccine formulation, were not signiﬁcantly different whether they were measured by an elution ELISA (avidity index; 0.9 ± 0.2 versus 1.1 ± 0.2 for CpG/Al(OH)3 and Al(OH)3, respectively, p > 0.05), or a competition ELISA (IC50, Table 1). It is possible that this resulted from the relatively low sensitivity of the ELISA assay used to measure afﬁnity in this study and that better differentiation may be achieved with more sensitive methods such as radioimmunoassay or Luminex based methods. The function of the anti-nicotine Abs in plasma recovered from NHPs, as determined by measuring the nicotine binding capacity in an equilibrium dialysis assay, showed that the nicotine binding capacity was signiﬁcantly greater with the CpG/Al(OH)3 adjuvanted vaccine than with the Al(OH)3 adjuvanted vaccine (p = 0.001); indeed every animal in the CpG/Al(OH)3 group had a greater nicotine binding capacity than the best binding capacity obtained in the Al(OH)3 group (Table 1). 3.3. Effect of anti-carrier antibodies in NHPs Anti-DT antibodies developed through immunization with the 3′ AmNic-DT conjugate and these were signiﬁcantly greater with CpG/ Al(OH)3 than Al(OH)3 (Fig. 4B). The presence of pre-existing anti-carrier antibodies did not prevent a boosting effect on anti-nicotine antibodies with subsequent doses of vaccine, and there was no evidence that the presence of anti-DT Ab were deleterious. For example, there was no correlation between anti-DT antibody levels immediately prior to the fourth
**** *** 60
Brain nicotine (ng-eq/g)
Fig. 3. Brain and blood levels of nicotine in mice. BALB/c mice (n = 10/gp) were immunized by IM injection with 10 μg of 3′AmNic-DT adjuvanted with Al(OH)3 (40 μg Al3+) ± CpG (50 μg) on days 0, 28 and 42. On day 56, animals received an IV injection of 3H-nicotine (0.05 mg/kg) and plasma and brains collected. Panel A shows nicotine levels in plasma (ng–eq/mL), and Panel B shows nicotine levels in brain (ng–eq/g). ⁎⁎⁎ and ⁎⁎⁎⁎ represent p b 0.001 and 0.0001, respectively.
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Anti-nicotine IgG Titer
p < 0.05 for all time points after 4 wks 2
Anti-DT IgG (GMT)
105 104 103 102 101 100 0
C Anti-DT IgG (log10) Pre-dose 4
r 2 =0.15
5 10 15 20 25 30 35 40 45 50
Anti-Nic-fold increase with dose 4 Fig. 4. Anti-nicotine antibody kinetics and avidity in non-human primates. Cynomolgus monkeys (n = 5/gp) were immunized by IM injection 3′AmNic-DT (100 μg) adjuvanted with Al(OH)3 (400 μg Al3+) ± CpG (500 μg) at the start of weeks 0, 4, 8, 27 and 40. Plasma was collected at various time-points and levels of anti-nicotine IgG (Panel A) and anti-DT IgG (Panel B) determined by ELISA. Panel C shows correlation between anti-DT levels at time of 4th immunization and fold increase in anti-nicotine Ab levels as result of 4th immunization.
immunization and the fold increase in anti-nicotine antibody levels induced by the fourth immunization (r2 = 0.15) (Fig. 4C). 4. Discussion In recent years, there have been a number of clinical studies conducted to evaluate the efﬁcacy of anti-nicotine vaccines [5,23–25].
The greatest clinical experience has been with NicVax, which unfortunately failed to show enhanced long-term quit rates compared to placebo treatment in phase 2 and phase 3 efﬁcacy trials. While the phase 2 proof-of-concept study with NicVax failed in the intentto-treat population, results were encouraging in that subjects in the 70th percentile for Ab responses had a greater long term abstinence rate than those receiving placebo . The subsequent phase 3 studies were designed to induce higher levels of Ab in a greater percentage of subjects via increased number of doses (6 instead of 4 or 5) and use of the highest antigen dose tested in phase 2 [5,24]. Unfortunately, this strategy did not result in improved clinical outcomes. The lack of clinical efﬁcacy despite high anti-nicotine Ab titers suggests that either the Abs were of inadequate function, for example due to low afﬁnity, or that the hypothesis for the mechanism of action for anti-nicotine vaccines is ﬂawed. Chronic smoking is associated with nicotine addiction, but it is also associated with complex psychosocial factors, which in theory could make quitting difﬁcult even in the complete absence of nicotine from the brain. However, it is known that low levels of nicotine, such as those resulting from nicotine replacement therapy, can aid smoking cessation through reduced craving without inducing reward, despite the presence of these psychosocial factors . This suggests that if enough nicotine can be kept from the brain by anti-nicotine antibodies, at least some clinical beneﬁt should be realized. This brings us back to the possibility that the quality of the antibodies induced by NicVax was insufﬁcient to have a meaningful clinical outcome. It is well known that a strong humoral response depends on both the quantity (titer) as well as quality (afﬁnity) of the Ab,  and several studies have now demonstrated the importance of both antibody titer and afﬁnity in determining anti-nicotine antibody function [28–30]. Antibody afﬁnity is largely determined by the antigen, so it is possible that the conjugate used in NicVax does not induce Ab of sufﬁciently high afﬁnity. The results of the present study emphasize that a high anti-nicotine antibody titer is not sufﬁcient to generate a good functional response. Using a model antigen that contains a different carrier, but the same hapten as NicVax, thus presenting nicotine to the immune system in a similar way, high titers of anti-nicotine Abs were induced in mice and NHP. Despite reasonably high titers of anti-nicotine antibodies (up to 105 in mice, 104 in NHP), 3′AmNic-DT adjuvanted with Al(OH)3 did not have meaningful functional outcomes. Namely, the amount of nicotine entering the brains of mice was not signiﬁcantly less than untreated controls and anti-nicotine Abs in NHP plasma bound less than 3% of the nicotine added to the equilibrium dialysis assay at a physiologically relevant level. Functional results were signiﬁcantly improved with the addition of CpG, apparently due to higher Ab titers in both mice and NHP, and also higher Ab afﬁnity in mice. Even then, nicotine entering the brain of immunized mice was reduced by only 30%; if similar results were realized in humans, the 70% of nicotine still entering the brain would likely be enough to activate reward mechanisms. Similarly, the functional readout for NHP samples showed binding of only about 30% of nicotine at 100 ng/mL, a level representative of high levels in arterial blood of a chronic smoker,  meaning 70% of the nicotine would be unbound and free to enter the brain. Our functional assessments in mice and NHP, which gave very similar results despite different assay approaches, are in line with earlier reports whereby similar reductions in brain levels were observed despite signiﬁcant nicotine sequestration in the plasma [32,33]. More recent studies have shown that changing hapten structure, linker composition, linker position and carrier protein can lead to much better functional outcomes, as determined by measuring brain levels of nicotine [7–11]. It is possible that better clinical outcomes in smoking cessation may be realized in the future with anti-nicotine vaccines containing a conjugate antigen optimized to induce not only high anti-nicotine Ab titers, but also Abs of high afﬁnity. But even then, adjuvants will be required and it is probable that an adjuvant combination such as
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Table 1 Immune responses in NHPs following 4 doses of 3′AmNic-DT with Al(OH)3 or Al(OH)3/CpG. Immunization
Anti-nicotine Ab titera
Anti-DT Ab titera
3′Am-Nic-DT + Al(OH)3
8001 8002 8003 8004 8005
2090 817 1470 6080 814 1655 ± 985 18,980 24,000 19,800 140,000 18,600 29,796 ± 23,950 0.0007
464 61 764 2590 34 286 ± 472 13,030 151,300 146,600 33,500 17,970 44,474 ± 31,410 0.0008
900 1105 1080 412 2553 1025 ± 358 807 1028 1224 977 1489 1081 ± 117 0.87
7.9 2.9 1.0 3.4 1.0 2.4 ± 1.3 14.8 39.0 17.8 67.5 20.2 26.9 ± 9.86 0.001
Geometric mean ± SEM 3′Am-Nic-DT + Al(OH)3/CpG
Geometric mean ± SEM Signiﬁcance (p value) a
9001 9002 9003 9004 9005
Values reported are average values of assay conducted on at least 2 separate occasions.
CpG and Al(OH)3 could further increase Ab function through its enhancement of Ab titer and afﬁnity. The combination of CpG and Al(OH)3 has not been previously reported with an anti-nicotine vaccine, although it was tested with an anti-heroin vaccine in mice, and surprisingly both antibody titer and afﬁnity were reported to be poorer than obtained with the same antigen adjuvanted with Al(OH)3 alone. Based on those results, the authors suggested that the adjuvant combination of CpG and Al(OH)3 was not suitable for hapten-conjugate antigens, possibly because of the Th1 biased immune responses that resulted . Our results with the 3′AmNic-DT vaccine show that this is clearly not the case because addition of CpG to the Al(OH)3-adjuvanted vaccine induced signiﬁcantly better anti-nicotine responses (antibody titer, afﬁnity and/or function) in mice and NHP, despite the fact that responses were shown to be more Th1 in mice. Anti-nicotine vaccines are expected to be administered as a series of several doses in order to induce sufﬁciently strong responses for sufﬁcient duration. This raises a potential issue of immunity against the carrier protein, which in the case of our model antigen, might be present even at prime since DT and related compounds are routinely used for vaccination against diphtheria or as conjugate carriers. With other conjugate vaccines, it has been reported that anti-carrier responses can have either positive or negative effects on vaccine responses, depending on the vaccine evaluated . In the present study, immunization of NHP with 3′AmNic-DT antigen, resulted in antibodies being raised against both nicotine and DT, especially when adjuvanted with CpG/Al(OH)3. However, even in the presence of high levels of anti-DT antibodies, anti-nicotine responses did not appear to be adversely affected as boosting effects did not correlate inversely with anti-DT responses. It is not possible to state that anti-DT antibodies had no effect on responses to subsequent boosts, but they did not preclude the boosting of waning responses back to peak or near-peak levels. In summary, we have shown that adjuvant combinations such as Al(OH)3 and CpG can enhance the function of anti-nicotine vaccines through augmenting both antibody titer and afﬁnity. While the results with the 3′AmNic-DT antigen are not overly encouraging for overcoming the issues of lack of clinical beneﬁt, similar adjuvant effects might be expected with an improved antigen optimized for inducing high afﬁnity Abs. To achieve this, it will likely be necessary to understand and optimize the protein carrier, the nicotine-like hapten and the relationship between the two. Indeed, a number of studies have now demonstrated that appropriate hapten design, including the use of different linkers and sites of attachment can result in enhanced functional responses in small animal models [27,29,30]. We have also now tested a series of nicotine-conjugate antigens in mice and demonstrated that immune responses, in particular antibody avidity, can be greatly inﬂuenced by both the hapten and linker
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