Novel Anti-Nicotine Vaccine Using a Trimeric Coiled-Coil Hapten Carrier

Abstract

Tobacco addiction represents one of the largest public health problems in the world and is the leading cause of cancer and heart disease, resulting in millions of deaths a year. Vaccines for smoking cessation have shown considerable promise in preclinical models, although functional antibody responses induced in humans are only modestly effective in preventing nicotine entry into the brain. The challenge in generating serum antibodies with a large nicotine binding capacity is made difficult by the fact that this drug is non-immunogenic and must be conjugated as a hapten to a protein carrier. To circumvent the limitations of traditional carriers like keyhole limpet hemocyanin (KLH), we have synthesized a short trimeric coiled-coil peptide (TCC) that creates a series of B and T cell epitopes with uniform stoichiometry and high density. Here we compared the relative activities of a TCC-nic vaccine and two control KLH-nic vaccines using Alum as an adjuvant or GLA-SE, which contains a synthetic TLR4 agonist formulated in a stable oil-in-water emulsion. The results showed that the TCC's high hapten density correlated with a better immune response in mice as measured by anti-nicotine Ab titer, affinity, and specificity, and was responsible for a reduction in anti-carrier immunogenicity. The Ab responses achieved with this synthetic vaccine resulted in a nicotine binding capacity in serum that could prevent >90% of a nicotine dose equivalent to three smoked cigarettes (0.05 mg/kg) from reaching the brain.

Figure 1. Structure of the Trimeric Coiled Coil protein.

(A) The amino acid sequence of each amphipathic peptide is comprised of 5 heptad repeats containing isoleucine at the “a” and “d” positions to form the hydrophobic interior of the trimer and lysine, arginine, and glutamic acid at the remaining positions. The carboxyl sequences of the CD4 T cell epitope are not represented. The position of each residue within the heptad repeat following self-assembly of the trimeric protein is represented by the three helical wheel projections. Forty eight lysine residues (yellow) are solvent exposed and available for hapten conjugation. Isoleucines (blue) comprise the hydrophobic core and arginine and glutamic acid residues (green) form salt bridges that stabilize the trimeric structure. (B) Homology model of the TCCnic peptide with rendered atoms represented by space filling Van der Waals radii. The model on the left is a view down the symmetrical axis of the trimer, whereas the right-hand model is a longitudinal view. The C-terminal portion contains CD4 T-cell epitopes (grey spheres) required for B-cell help. Nicotine-6-hexanoic acid haptens (black bonds) were manually added to the ε-amino group of 12 randomly chosen lysine residues using the Molefacture plug-in from the Visual Molecular Dynamics software package. Further details of model construction are provided in the “Materials and Methods” section.

Figure 2. A comparison of the relative size and lysine content of the TCC and four hapten carriers; KLH, Exotoxin A (ExoT A), Tetanus toxoid (Tet T), and the inactivated diphtheria toxin protein CRM197; (A) The number of total amino acids with the number of lysines available for hapten conjugation reported above the bar; (B) The percentage of lysines in each carrier protein.

Figure 3. Anti-nicotine antibody responses in immunized mice.

C57BL/6 mice (5/grp) were injected (d0, d14, d131) with either PBS or 2.5 µg of the indicated conjugate hapten carriers and adjuvants, and sera was assayed for anti-nicotine Ab titers by ELISA. TCCnic-12 contained an average 12 haptens per trimer, whereas KLHnic-22 and KLHnic-100 contained, respectively an average 22 and 100 haptens per monomer protein. Comparisons between groups were conducted by unpaired two-tailed t-test; *p<0.004; **p<0.002.

Figure 4. Day 160 anti-nicotine Ab titers.

C57BL/6 mice (5/grp) were immunized (d0, d14, d146) with either PBS or 2.5 µg of the indicated conjugated hapten carriers and adjuvants, and serum was assayed by ELISA. Comparisons between groups were conducted by unpaired two-tailed t-test; *p<0.003; **p<0.0001.

Figure 5. Vaccine dose response.

C57BL/6 mice (5/grp) were immunized with the indicated doses of TCCnic-12 in the absence (A) and presence (B) of GLA-SE, and d35 serum was assayed for anti-nicotine Ab by ELISA.

Figure 6. Anti-carrier Ab responses in immunized mice.

C57BL/6 mice (5/grp) were injected (d0, d14) with the indicated carriers in the presence of GLA-SE. TCCnic-2, TCCnic-12, and TCCnic-42 contained, respectively, an average 2, 12, and 42 haptens per trimer. Day 28 serum from each group was assayed for Ab that bound the corresponding unconjugated hapten carrier.

Figure 7. Antibody specificity.

The specificity of nicotine binding to antisera collected from TCCnic-12 immunized mice was determined by competitive ELISA for nicotine, cotinine and acetylcholine. IC₅₀ values for cotinine were 1000-fold greater than nicotine and could not be calculated for acetylcholine due to a lack of inhibition.

Figure 8. Relative affinities of anti-nicotine Abs induced by TCCnic-12, KLHnic-22, and KLHnic-100, either alone or in the presence of Alum or GLA-SE.

C57BL/6 mice (5/grp) were injected (d0, d14, d146) with either PBS or 2.5 µg of the indicated conjugate hapten carriers and adjuvants. The geometric mean Kd values indicated above each data set were determined by competitive ELISA.

Figure 9. Serum nicotine binding capacity was determined by measuring bound and free concentrations of nicotine at equilibrium.

Kd values (Fig. 8) were used to calculate total antibody concentrations according to the law of mass action equation: Kd  =  [Nic][IgG]/[Nic-IgG]. Comparisons between groups were conducted by unpaired two-tailed t-test; *p<0.04; **p<0.01; ***p<0001.

Figure 10. Anti-nicotine Ab function in mice.

C57BL/6 mice (5/grp) were injected (d0, d14, d146) with either PBS or 2.5 ug of the indicated conjugate hapten carriers and adjuvants. Mice (5/grp) were injected on d160 with a dose of nicotine tartrate equivalent to 3 cigarettes (1.2 ug). Five minutes later the mice were sacrificed, tissues removed and the amounts of nicotine in brain (A); and serum (B) were measured by mass spectrometry; * p<0.05; ** p<0.007; *** p<0.0003.