Enhancing immunogenicity of a 3′aminomethylnicotine-DT-conjugate anti-nicotine vaccine with CpG adjuvant in mice and non-human primates

https://doi.org/10.1016/j.intimp.2013.03.021Get rights and content

Highlights

  • Adjuvant combination was studied to enhance nicotine vaccine efficacy.

  • Antibody titer was higher in mice and NHPs with CpG/Al(OH)3 than with Al(OH)3 alone.

  • Antibody function was also higher with CpG/Al(OH)3 than with Al(OH)3 alone.

Abstract

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.

Introduction

Tobacco use remains the world's leading cause of preventable death, killing nearly six million people annually [1]. 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, 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 [4]. 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 [5] and NicVax [6] 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 the curve of antibody titers from the time of the first dose until 26 weeks for NicVax,[6] or from 3 months to 6 months for NicQb [5] 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], [8], [9], [10], [11], [12], [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 [14]. 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], [16], [17], [18] and, in one study where it was evaluated, also significantly enhanced antibody affinity[19].

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.

Section snippets

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),

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 < 0.05) (Fig. 1A). In addition, antibody affinity was shown to be significantly increased by CpG (p < 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 < 0.01), which in mice is indicative of a more Th1-biased response

Discussion

In recent years, there have been a number of clinical studies conducted to evaluate the efficacy of anti-nicotine vaccines [5], [23], [24], [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 intent-to-treat population, results were encouraging in that subjects in the 70th percentile for

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    Present address: Health Protection Agency, Process and Analytical Development Group, Porton Down, Salisbury, UK.

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