Structurally distinct nicotine immunogens elicit antibodies with non-overlapping specificities

Abstract

Nicotine conjugate vaccine efficacy is limited by the concentration of nicotine-specific antibodies that can be reliably generated in serum. Previous studies suggest that the concurrent use of 2 structurally distinct nicotine immunogens in rats can generate additive antibody responses by stimulating distinct B cell populations. In the current study we investigated whether it is possible to identify a third immunologically distinct nicotine immunogen. The new 1'-SNic immunogen (2S)-N,N'-(disulfanediyldiethane-2,1-diyl)bis[4-(2-pyridin-3-ylpyrrolidin-1-yl)butanamide] conjugated to keyhole limpet hemocyanin (KLH) differed from the existing immunogens 3'-AmNic-rEPA and 6-CMUNic-BSA in linker position, linker composition, conjugation chemistry, and carrier protein. Vaccination of rats with 1'-SNic-KLH elicited high concentrations of high affinity nicotine-specific antibodies. The antibodies produced in response to 1'-SNic-KLH did not appreciably cross-react in ELISA with either 3'-AmNic-rEPA or 6-CMUNic-BSA or vice versa, showing that the B cell populations activated by each of these nicotine immunogens were non-overlapping and distinct. Nicotine retention in serum was increased and nicotine distribution to brain substantially reduced in rats vaccinated with 1'-SNic-KLH compared to controls. Effects of 1'-SNic-KLH on nicotine distribution were comparable to those of 3'-AmNic-rEPA which has progressed to late stage clinical trials as an adjunct to smoking cessation. These data show that it is possible to design multiple immunogens from a small molecule such as nicotine which elicit independent immune responses. This approach could be applicable to other addiction vaccines or small molecule targets as well.

Figure 1

Nicotine and hapten structures. The 1′-SNic disulfide dimer was reduced to monomer prior to conjugation.

Figure 2

1% Agarose gel of unconjugated maleimide-activated KLH (outer lanes) and 1′-SNic-KLH synthesized using a 700:1 molar hapten:protein ratio (middle lane) showing a change in mobility with conjugation.

Figure 3

Efficacy of 1′-SNic-KLH synthesized using a range of conjugation reaction molar hapten:protein ratios. Top, nicotine-specific antibody titers (mean±SD) in vaccinated rats. Middle, retention of nicotine in serum; Bottom, nicotine distribution to brain 3 minutes after i.v. administration of 0.03 mg/kg nicotine (n=3 per group except n=6 for 700:1–25 ug group). Efficacy increased up to a reaction molar ratio of 700:1 but not with higher ratios.

Figure 4

Ligand cross reactivity of antiserum from rats vaccinated with 1′-SNic-KLH determined by competition ELISA.

Figure 5

Immunogen cross reactivity ELISA assay. Antisera from vaccinated rats were plated against nicotine haptens conjugated to various carrier proteins. Panel titles indicate the antiserum and horizontal axis labels indicate the coating antigen. Titers are expressed as % of control with 100% (dark bars) representing the titer obtained for each immunogen plated against the corresponding coating antigen containing the same hapten. See Table 1 for corresponding ELISA titers expressed as absolute values.

Figure 6

Nicotine retention in serum and distribution to brain in vaccinated rats (mean±SD, n=6 per group). The 1′-Snic-KLH immunogen was as effective as the other immunogens. * p <0.05 compared to control.

Scheme 1

Synthesis of 1′-SNic and conjugation to the carrier protein. a) 2-[(triphenylmethyl)sulfanyl]ethan-1-amine hydrochloride, BOP, TEA, THF; b) Hg(OAc) ₂, H₂S(g), EtOH; c) TCEP, 1′-SNic, maleimide activated BSA, PBS.