Immunization to nicotine with a peptide-based vaccine composed of a conformationally biased agonist of C5a as a molecular adjuvant

Abstract

This paper describes the synthesis of a nicotine hapten (Nic) that possesses a carboxyl sidearm functional group allowing for conjugation to a peptide via amide bond formation. Nic was attached to the N-terminal amino group of a 19-residue peptide composed of a conformationally biased agonist of human C5a (YSFKPMPLaR), which is used as a molecular adjuvant and a B cell epitope of human MUC1 glycoprotein (YKQGGFLGL) to yield a peptide-based nicotine vaccine, NicYKQGGFLGLYSFKPMPLaR. Rats immunized with this vaccine were significantly less sensitive to behavioral effects (a Pavlovian discrimination task) induced by their exposure to high concentrations of nicotine (0.4 mg/kg) relative to their non-vaccinated counterparts. The attenuation of these nicotine-induced behavioral effects emanated from the presence of nicotine-specific antibodies (Abs) that were present in the sera of vaccinated rats even after their repeated exposure to high concentrations of nicotine during the time required to perform the behavioral assays. These results suggest that immunization with NicYKQGGFLGLYSFKPMPLaR in the absence of adjuvant is an effective means of inducing a nicotine-specific Ab response, which is capable of attenuating nicotine-induced behavioral/psychoactive effects.

Introduction

The disease consequences of habitual tobacco smoking are irrefutable and impose an enormous public health burden on the world's population. As well established as the adverse health consequences of smoking, so too are the health benefits derived from smoking cessation. Quitting from smoking, however, can be difficult and even with support, only a third of the smoking population is able to quit [1]. Since tobacco dependence results in large part from an addiction to nicotine [2], [3], strategies aimed at smoking cessation have targeted nicotine's addictive effects. The mainstay of these treatments is nicotine replacement therapy with gradually decreasing doses of nicotine delivered via transdermal patches or inhaled formulations. Although these approaches have proven beneficial to some, the continued administration of the principal addictive substance in tobacco smoke is not an ideal approach to smoking cessation and the nicotine itself may exert adverse cardiovascular effects. Nicotine addiction also is treated with neuroreceptor antagonists/partial agonists such as mecamylamine and buproproin [4] in order to modulate certain neurotransmitter pathways. Since these receptors and signaling pathways also mediate normal brain function, side effects are common with the use of these drugs. Thus, there is a growing need for the development of improved strategies to help individuals quit smoking and maintain long-term compliance in an effort to prevent the costly and debilitating diseases caused by smoking.

An innovative approach to smoking cessation is to treat the addictive properties of nicotine via immunization with a nicotine vaccine. The rationale is that antibodies (Ab) generated in response to the vaccine would bind nicotine in peripheral circulation and reduce access to receptor sites in the brain where it induces its addictive effects. This peripheral site of action, along with the high specificity and affinity of nicotine Abs, makes vaccination an attractive therapeutic approach to smoking cessation. Also, vaccination has the potential of inducing a memory response, wherein anti-nicotine immunity may be invoked when one is exposed to nicotine, an attractive feature for ensuring long-term compliance. An appropriately designed nicotine vaccine would be less expensive than conventional treatments and would be particularly beneficial to those who do not respond to conventional nicotine addiction regimens or who cannot tolerate the side effects from the drugs used in these treatments.

The therapeutic potential of vaccination for smoking cessation has been demonstrated by reports showing that rats immunized with vaccines made by conjugating many nicotine haptens to a carrier protein (i.e., an immunoconjugate) admixed in adjuvant, generated high titers of nicotine-specific Abs in serum. Moreover, immunized rats were significantly less sensitive to nicotine-induced behavioral and physiologic effects than their non-vaccinated counterparts [5], [6], [7], [8], [9], [10]. This therapeutic effect emanated from the ability of nicotine-specific Abs generated in response to the immunoconjugate vaccine to bind nicotine in peripheral circulation, which reduced its distribution to the brain.

In this paper, we describe the design and synthesis of a peptide-based nicotine vaccine that utilizes a conformationally biased, response-selective agonist of C5a (YSFKPMPLaR) as a molecular adjuvant. We have shown YSFKPMPLaR to be an effective molecular adjuvant in inducing robust, antigen (Ag)-specific humoral (Ab), and cell-mediated immune responses to various peptide epitopes covalently attached to its N-terminus [11], [12], [13]. This molecular adjuvant property of YSFKPMPLaR results from the unique conformational features that are biased in the peptide [14], which we have shown are significantly better accommodated by C5a receptors (C5aR) expressed on antigen presenting cells (APC) than C5aRs on inflammatory neutrophils [15]. YSFKPMPLaR, therefore, delivers both the attached epitope/Ag and stimulatory signals to C5aR-bearing APCs. As a result, Ag-specific immune response can be generated in the absence of added adjuvant and, consequently, with little or no inflammatory side effects [13].

Herein, we report the immunologic results of rats vaccinated in the absence of adjuvant with a peptide-based, YSFKPMPLaR-containing vaccine in which a single nicotine hapten is used as the target Ag. These results are discussed in terms of the unique molecular adjuvant properties of YSFKPMPLaR, which serve to underscore its therapeutic potential in the design of peptide-based vaccines for drugs of addiction.