Optimization of a multivalent peptide vaccine for nicotine addiction

Abstract

We have been optimizing the design of a conjugate vaccine for nicotine addiction that employs a peptide-based hapten carrier. This peptide, which is produced by solid-phase protein synthesis, contains B cell and T cell epitope domains and eliminates the non-relevant, but highly immunogenic sequences in microbial carriers. In this report, the amino acid sequences in the T cell domain were optimized for improved vaccine activity and multivalent formulations containing structurally distinct haptens were tested for the induction of additive antibody responses. Trivalent vaccines produced antibody concentrations in mice that were 100 times greater than the amount of nicotine measured in smokers, and significantly reduced acute nicotine toxicity in rats. Two additional features were explored that distinguish the peptide from traditional recombinant carriers. The first is the minimal induction of an anti-carrier response, which can suppress nicotine vaccine activity. The second employs solid-phase synthesis to manufacture haptenated peptide. This approach obviates conventional conjugation chemistries and streamlines production of a more potent vaccine antigen.

Keywords: Addiction; Antibody response; Hapten; Nicotine vaccine; Solid-phase protein synthesis.

Fig. 1.

Nicotine haptens used in this study (see Methods).

Fig. 2.

TCE fusions synergistically improve Ab responses. Hapten 1’ was conjugated to P9, P12, or P13 (Table 1). Mice (n=10/grp) were immunized with 5 μg peptide plus adjuvant. Serum was collected on d56 and assayed for anti-nicotine Ab titers (A) and relative nicotine avidities (B). Comparisons between groups were conducted by ANOVA. *p<0.001.

Fig. 3.

P10 carrier selection. Peptides P9, P10, P14 and P15 (Table 1) were conjugated to hapten 3’ and mice were immunized with 5 μg of each peptide plus adjuvant. Ab titers (A) and relative Ab avidities (B) were assayed by ELISA using d56 sera. Comparisons between groups were conducted by ANOVA. *p<0.05.

Fig. 4.

P10 peptide induces dramatically lower anti-carrier Ab titers as compared to CRM₁₉₇. Mice (n=8) were immunized with 10 μg of either 1’-P10 or 1’-CRM₁₉₇ adjuvanted with GLA-SE. Anti-nicotine Ab titers (A), relative avidities (B), and anti-carrier Ab titers were assayed by ELISA using d56 sera. Comparison between groups were conducted by unpaired t-test. *p<0.00001

Fig. 5.

Hapten activity comparison. The P10 peptide was conjugated to the indicated haptens, formulated with adjuvant, and then injected (2 μg) into mice. Day 56 sera was used to determine Ab titers (A), relative avidities (B), and nicotine binding capacities (C).

Fig. 6.

Multivalent nicotine vaccines induce additive Ab responses. Monovalent, bivalent and trivalent vaccines were formulated with the indicated conjugates (5 μg each) plus adjuvant and then injected into mice (10/grp). Day 56 serum was used to determine average nicotine binding capacities per group.

Fig. 7.

Behavioral responses in rats following a nicotine challenge. Baseline mobility was measured in immunized and age-matched control rats after injection with PBS. One week later, animals were injected (i.p.) with 3.0 mg/kg nicotine, returned to chambers and measured in two ways (see Methods). (A) Symptoms of nicotine toxicity using a 5-point scoring system (see Methods). Comparisons between groups were conducted by unpaired t-test. *p=0.006. (B) Horizontal distance traveled. Three comparisons are shown: control rats injected with PBS or nicotine, vaccinated rats injected with PBS or nicotine, and control versus immunized rats following nicotine injection. Comparisons between groups were conducted by unpaired t-test. For the control comparison between baseline and nicotine treatment, *p=0.0001. For nicotine-treated control vs vaccinated groups, *p=0.016

Fig. 8.

Nicotine pharmacokinetics in control and vaccinated rats. Animals were injected (i.p.) with 0.3 mg/kg nicotine and tissues collected 5 min later. Nicotine levels were measured in brain and serum by LC/MS. Comparisons between groups were conducted by unpaired t-test. *p<0.001.

Fig. 9.

Peptides synthesized with hapten building blocks induce superior Ab responses relative to conjugated peptides. (A) Structure of the hapten 6-lysine building block with an Fmoc protected side chain (outlined). (B) Peptide sequence showing building block insertion points (arrows). P10 peptides were synthesized with one BB positioned at the amino terminal location, BBs at all three sites or conjugated by conventional methods with an average 3.25 haptens per peptide. (C) CD-1 mice (n=8) were immunized on days 0, 21 and 42 with 5 μg adjuvanted peptide and day 56 serum was assayed for Ab titer, avidity and average nicotine binding capacity. Comparisons between groups were conducted by unpaired t-test. *p<0.008.