Enhanced immunogenicity of a bivalent nicotine vaccine

Abstract

The efficacy of nicotine vaccines for smoking cessation is dependent upon their ability to elicit sufficiently high serum antibody concentrations. This study compared two nicotine immunogens representing different hapten presentations, 3′-aminomethyl nicotine conjugated to recombinant Pseudomonas exoprotein A (3′-AmNic-rEPA) and 6-carboxymethlureido nicotine conjugated to keyhole limpet hemocyanin (6-CMUNic-KLH), and assessed whether their concurrent administration would produce additive serum antibody concentrations in rats. Effects of vaccination on nicotine pharmacokinetics were also studied. Vaccination of rats with these immunogens produced non cross-reacting nicotine-specific antibodies (NicAb). Serum NicAb concentrations elicited by each individual immunogen were not affected by whether the immunogens were administered alone as monovalent vaccines or together as a bivalent vaccine. The total NicAb concentration in the bivalent vaccine group was additive compared to that of the monovalent vaccines alone. Higher serum NicAb concentrations, irrespective of which immunogen elicited the antibodies, were associated with greater binding of nicotine in serum, a lower unbound nicotine concentration in serum, and lower brain nicotine concentration. These results demonstrate that it is possible to design immunogens which provide distinct nicotine epitopes for immune presentation, and which produce additive serum antibody levels. The concurrent administration of these immunogens as a bivalent vaccine may provide a general strategy for enhancing the antibody response to small molecules such as nicotine.

Introduction

Nicotine is the principal addictive component of tobacco smoke [1]. Vaccination against nicotine is a potential means of modifying or attenuating nicotine's addictive effects [2], [3]. Vaccination of rats with a suitable nicotine immunogen elicits the production of nicotine-specific antibodies which bind nicotine in serum and extracellular fluid, reduce the concentration of unbound nicotine, reduce or slow nicotine distribution to the brain [4], [5], [6], [7], and attenuate a variety of nicotine-induced behaviors in rats including locomotor activation [8], nicotine discrimination [9], and the acquisition, maintenance and reinstatement of nicotine self administration [10], [11]. The effects of immunization on nicotine pharmacokinetics in rats are a function of both the serum NicAb concentration and antibody affinity for nicotine. Close correlations have been found between the serum NicAb concentration and the percent of nicotine bound in serum, the concentration of unbound nicotine in serum, and the distribution of nicotine to brain [5], [12], [13]. Recent studies also show a correlation between the serum antibody concentration and a behavioral outcome, locomotor sensitization to nicotine (unpublished data). Three nicotine vaccines are in Phases II–III clinical trials as adjuncts for smoking cessation and have shown preliminary evidence of efficacy. In each of these clinical trials, efficacy has been closely related to the serum antibody concentration or titer in that increased smoking cessation rates were observed in only those subjects with the highest serum NicAb levels [4], [14], [15], [16], [17](C. Bunce, personal communication). These highly congruent animal and human data clearly demonstrate the need to achieve high serum NicAb concentrations in order to maximize the efficacy of vaccination for treating tobacco dependence.

Two challenges have emerged from clinical trials regarding the generation of high antibody concentrations from nicotine vaccines. First, the mean serum NicAb concentrations reported have been modest, 32 ug/ml in one trial [14], which is considerably lower than typical levels of 180–250 ug/ml in rats vaccinated with the same immunogen [5], [18]. This difference may be due in part to the ability to use Freund's adjuvant in rats, while alum was used in humans. Second, considerable variability in serum antibody concentrations has occurred following vaccination against nicotine; a 30-fold range in one study, from a high of 100 µg/ml to less than10 µg/ml [14]. Although only limited data have been published, these challenges appear to be common to all three nicotine vaccines currently under clinical investigation [14], [15](C. Bunce, personal communication). As a result, only a minority of vaccinated subjects have achieved antibody levels sufficient to enhance smoking cessation rates.

The current study investigated the combined administration of two distinct nicotine immunogens in rats as a means of enhancing the total serum NicAb response, and also reducing individual variability. The two immunogens utilized different linker positions, linker composition, and carrier proteins in order to provide distinct hapten presentations. It was hypothesized that immune responses to the two immunogens would be independent so that combining the two immunogens into a bivalent vaccine would not compromise the immune response to each individual immunogen, and would produce additive serum antibody levels. The effects of these vaccines on the distribution and serum protein binding of a single nicotine dose were also studied.