Prescreening of Nicotine Hapten Linkers in Vitro To Select Hapten-Conjugate Vaccine Candidates for Pharmacokinetic Evaluation in Vivo

Article abstract of DOI:10.1021/acscombsci.6b00179

Since the demonstration of nicotine vaccines as a possible therapeutic intervention for the effects of tobacco smoke, extensive effort has been made to enhance nicotine specific immunity. Linker modifications of nicotine haptens have been a focal point for improving the immunogenicity of nicotine, in which the evaluation of these modifications usually relies on in vivo animal models, such as mice, rats or nonhuman primates. Here, we present two in vitro screening strategies to estimate and predict the immunogenic potential of our newly designed nicotine haptens. One utilizes a competition enzyme-linked immunoabsorbent assay (ELISA) to profile the interactions of nicotine haptens or hapten-protein conjugates with nicotine specific antibodies, both polyclonal and monoclonal. Another relies on computational modeling of the interactions between haptens and amino acid residues near the conjugation site of the carrier protein to infer linker-carrier protein conjugation effect on antinicotine antibody response. Using these two in vitro methods, we ranked the haptens with different linkers for their potential as viable vaccine candidates. The ELISA-based hapten ranking was in an agreement with the results obtained by in vivo nicotine pharmacokinetic analysis. A correlation was found between the average binding affinity (IC₅₀) of the haptens to an anti-Nic monoclonal antibody and the average brain nicotine concentration in the immunized mice. The computational modeling of hapten and carrier protein interactions helps exclude conjugates with strong linker-carrier conjugation effects and low in vivo efficacy. The simplicity of these in vitro screening strategies should facilitate the selection and development of more effective nicotine conjugate vaccines. In addition, these data highlight a previously under-appreciated contribution of linkers and hapten-protein conjugations to conjugate vaccine immunogenicity by virtue of their inclusion in the epitope that binds and activates B cells.

Full text of DOI:10.1021/acscombsci.6b00179

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Article
Pre-screening of nicotine hapten linkers in vitro to select hapten-
conjugate vaccine candidates for pharmacokinetics evaluation in vivo
Viswanath Arutla, Joseph Leal, Xiaowei Liu, Sriram Sokalingam, Michael Raleigh,
Adejimi Adaralegbe, Li Liu, Paul R. Pentel, Sidney M. Hecht, and Yung Chang
ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.6b00179 • Publication Date (Web): 06 Apr 2017
Downloaded from http://pubs.acs.org on April 7, 2017
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Pre-screening of nicotine hapten linkers in vitro to select hapten-conjugate vaccine
candidates for pharmacokinetics evaluation in vivo
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Viswanath Arutla,#,† Joseph Leal,#,‡,^ Xiaowei Liu#,‡ Sriram Sokalingam, ‡ Michael Raleigh,§
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Adejimi Adaralegbe,‡ Li Liu,╤,↕ Paul R. Pentel,§ Sidney M. Hecht,*,†, and Yung Chang,*,‡,
⊥
∥
†Biodesign Center for BioEnergetics, ‡Biodesign Center for Immunotherapy, Vaccines and
Virotherapy, and ╤Biodesign Center for Personalized Diagnostics, Arizona State University,
Tempe, Arizona 85287, United States
↕Department of Biomedical Informatics, College of Health Solutions, Arizona State University,
Scottsdale, 85259, United States
§Minneapolis Medical Research Foundation, Minneapolis, Minnesota 55404, United States
∥School of Life Sciences, and ⊥Department of Chemistry and Biochemistry, Arizona State
University, Tempe, AZ 85287, United States
^ Current address: Department of Immunology, University of Washington, Seattle, Washington,
98109, United States
# These individuals contributed equally
* Coꢀcorresponding authors
Address for Yung Chang and Sidney Hecht: 1001 S. McAllister Ave, Tempe, AZ, 85287ꢀ7501
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ABSTRACT
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Since the demonstration of nicotine vaccines as a possible therapeutic intervention for the effects
of tobacco smoke, extensive effort has been made to enhance nicotine specific immunity. Linker
modifications of nicotine haptens have been a focal point for improving the immunogenicity of
nicotine, in which the evaluation of these modifications usually relies on in vivo animal models,
such as mice, rats or nonꢀhuman primates. Here, we present two in vitro screening strategies to
estimate and predict the immunogenic potential of our newly designed nicotine haptens. One
utilizes a competition enzymeꢀlinked immunoabsorbent assay (ELISA) to profile the interactions
of nicotine haptens or haptenꢀprotein conjugates with nicotine specific antibodies, both
polyclonal and monoclonal. Another relies on computational modeling of the interactions
between haptens and amino acid residues near the conjugation site of the carrier protein to infer
linkerꢀcarrier protein conjugation effect on antiꢀnicotine antibody response. Using these two in
vitro methods, we ranked the haptens with different linkers for their potential as viable vaccine
candidates. The ELISAꢀbased hapten ranking was in an agreement with the results obtained by in
vivo nicotine pharmacokinetic analysis. A correlation was found between the average binding
affinity (IC50) of the haptens to an antiꢀNic monoclonal antibody and the average brain nicotine
concentration in the immunized mice. The computational modeling of hapten and carrier protein
interactions helps exclude conjugates with strong linkerꢀcarrier conjugation effects and low in
vivo efficacy. The simplicity of these in vitro screening strategies should facilitate the selection
and development of more effective nicotine conjugate vaccines. In addition, these data highlight
a previously underꢀappreciated contribution of linkers and haptenꢀprotein conjugations to
conjugate vaccine immunogenicity by virtue of their inclusion in the epitope that binds and
activates B cells.
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INTRODUCTION
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Nicotine (Nic) vaccines have been explored as an intervention strategy to improve
smoking cessation. The rationale for this therapeutic strategy is based on the production of antiꢀ
Nic antibodies that could sequester Nic within the blood circulation and block its entry into the
brain, thereby reducing Nic dependence. However, the success of this intervention is dependent
on the the ability of the vaccine to generate high quality antiꢀNic antibody responses that produce
high levels of antibodies with highꢀbinding affinity to Nic. To date, the vaccines tested in clinical
trials or investigated in preclinical studies have failed to induce sufficient antibody responses to
enable their use as adjuncts for smoking cessation.1ꢀ2
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Given its small size, Nic is not immunogenic on its own. Like many small molecules, the
immunogenicity of Nic is realized by making linkerꢀcontaining Nic haptens (HPs) for their
conjugation to a carrier protein. The protein serves as sources of antigenic epitopes for engaging
T cells to induce Tꢀcell dependent antibody responses. To improve the immunogenicity of Nic
vaccines, various strategies have been attempted, ranging from linker modification, carrier
selection, conjugation optimization, adjuvant addition, and multivalent vaccine combination, to
nanoparticle packaging.1 Unlike the conformational rigidity of cocaine and heroin structures,
which are believed to confer good immunogenicity of these molecules, the flexibility of Nic
structure has been accounted for the poor immunogenicity of Nic HPs.3 To increase the Nic
immunogenicity, Janda and coworkers developed several strategies, including design of
conformationally constrained Nic analogues, use of multivalent scaffolds to increase HP density,
and isolation of enantiopure HPs, aiming to direct Nic epitopes to antigenꢀspecific B cells.4ꢀ6 In
addition, a comprehensive study conducted by Pryde et al.7, has surveyed various linkers that
differ in length, rigidity, and polarity, as well as the position of attachment of Nic to the linkers.
In this study, rigid linkers were found to reduce the immunogenicity of Nic diphtheria toxoid
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conjugates, as well as functional activities of elicited responses, which is consistent with the
finding reported by de Villiers, et al. 8 Yet, variations in linker length or polarity appeared to
exert minimal effect on the antibody level, affinity, or functionality of the antibody responses.7
Thus, it remains elusive as to what features of linker structures confer high quality antiꢀNic
antibody responses.
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Recently, the selection of appropriate Nic HPs has also been coupled to the enumeration
of HPꢀspecific naïve B cells present in the host.9 Using two different Nic HPs that are conjugated
to keyhole limpet hemocyanin (KLH), Laudenbach et al. reported that a higher number of HPꢀ
specific naïve (preꢀimmunization) B cells was correlated with a stronger antiꢀNic antibody
response elicited by that particular HPꢀKLH conjugate.9 Although profiling of HPꢀspecific naïve
B cells may facilitate the selection of optimal HPs for B cell engagement, this strategy is not well
suited to screening a large number of HPs, and may not serve to select those HPs that favour
antibody maturation toward high affinity and high specificity to free Nic.
Instead of examining Nic binding of naïve B cells present in animals, we posited that
antiꢀNic antibodies with high binding affinities for free Nic may serve as a surrogate for Nic
specific B cells, since the Nicꢀbinding specificity of these antibodies and the surface
immunoglobulins (Igs) expressed on the B cells are clonally related. This idea was inspired by
the success of “vaccine reverse engineering”, first demonstrated in the development of HIV
vaccine.10 The reverse engineering focuses on the interactions between neutralizing monoclonal
antibodies (mAbs) and antigens of an interest, which infers the structures of the epitopes that fit
into neutralizing mAbs.11 Essentially, neutralizing mAbs were used as a bait to search for and
capture mAbꢀspecific “immunogenic” epitopes. Based on this principle, we asked whether highꢀ
affinity Nicꢀbinding antibodies could be exploited as a screening bait to search for Nicꢀlike
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epitopes. Because free Nic itself is not immunogenic, antiꢀNic antibodies were actually raised
against Nic HPs (Nic moiety plus linker) conjugated to a protein carrier. Emphasis in the past has
been placed on designing HPs with nonꢀimmunogenic linker sequences (e.g. using linkers with
minimal structural complexity) in order to minimize the production of antibodies directed against
linker rather than Nic. However, while it is the binding of free Nic that is desired for therapeutic
purposes, Nicꢀspecific B cells are usually engaged by linkerꢀcontaining HPs for their activation.
We hypothesized that effective HPs would use both their Nic and their linker components to bind
and engage B cells while generated antibodies from these B cells could recognize free Nic as
well as the complete HP. Thus, according to the reverse engineering principle, by using a known
Nicꢀbinding antibody as a screening bait, we could search for those HPs that can best fit within
the Nicꢀbinding site of the antibody.
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AntiꢀNic antibodies can be distinguished by HP linker positions attached to the Nic
moiety, e.g., the antibodies raised against Nic HPs linked via 1’position do not bind HPs with
linkers attached to different Nic positions, like 3’ꢀaminomethylꢀNic conjugate and 6ꢀ
(carboxymethylureido)ꢀNic conjugate, respectively.12 Interstingly, although the antibodies raised
againt these different HPs that vary in linker attachment positions fail to crossꢀreact among these
different families of HPs, they can all bind to free Nic, which highlights the dualꢀbinding
features of Nicꢀspecific B cells, i.e., nicotine and linker attachment. Thus, the antibodyꢀbased
screening strategy could only be applied in HPs with the same linker attachment position as the
HP originally used to elicit the screening antibodies.
To test our hypothesis, we focused on new HPs with linkers attached to Nic at the 1’ꢀ
position employing the pyrrolidyl methyl group, since using this position for attachment has been
shown to allow generation of high affinity Nicꢀspecific antibodies, and preserves the chirality of
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Nic as the Sꢀ(ꢀ) enantiomer, the main form found in cigarette smoke.13 Using 1’Nic HP, that was
first reported by Janda,14 as our prototype, we introduced different degrees of linker
modifications and tested their binding activities to both polyclonal and monoclonal antiꢀNic
antibodies. Our strategy was to screen HPs by measuring their binding to Nicꢀspecific antibodies
via competition ELISA, in which the HPs serve as competitors for the binding of antibody to
wells coated with 1’Nic HP linked to a protein unrelated to the carrier protein. We recognize the
dilemma that the linker would not contribute to altering the pharmacokinetics of free Nic
although it affects the binding of the antibody to Nic epitope in the competition
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ELISA. Nevertheless, we reasoned that linker binding is not entirely irrelevant, nor is it
necessarily undesirable because it is a determinant of how well an immunogen containing this
HP will bind to B cell receptors having specificity similar to that of the screening antibody and
activate those B cells.
Besides HP linkers, the amino acids near the HPꢀprotein conjugation site could also
interact with conjugated Nic HPs and influence their access to the same antiꢀNic antibody,
possibly accounting for linkerꢀcarrier protein conjugation effect. To address this issue, we
applied computational molecular modeling to profile the display of Nic HPs on streptavidin, a
carrier protein, by analyzing haptenꢀstreptavidin (HPꢀSA) interactions near the conjugation site.
The goal of this modeling was to estimate the extent to which HPs would interact with adjacent
amino acids. Such an interaction may go both ways. On one hand, it may help orient and
constrain the Nic moiety for better engagement with Nicꢀspecific B cells. On the other hand, too
much interaction might bury the epitope, limit recognition by Nicꢀspecific B cells, and/or
possibly create new antigenic epitopes unrelated to Nic. Therefore, we hypothesized that high
HPꢀprotein interaction would interfere with the generation of Nicꢀfocused antibody responses.
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This effect could also be assessed by competition ELISAs to examine and compare the binding
activity of the antiꢀNic antibody to free HPs vs HPꢀSA conjugates that contain the same HPs.
The combination of computational modeling and competition ELISA of HPꢀSA conjugates was
intended to offer an independent evaluation of the steric accessibility of conjugated HPs to the
Nicꢀbinding sites of antiꢀNic antibodies.
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The predictions derived from these in vitro experimental analyses were tested through
immunization of mice and analysis of antibody effects on Nic distribution in serum and brain.
Here, we showed a good agreement between the in vitro binding of HP candidates to Nicꢀ
specific antibodies and nicotine pharmacokinetics demonstrated in vivo. In addition, our analyses
revealed a previously underꢀappreciated role of linkers and HPꢀprotein conjugations in
modulating the access of Nicꢀconjugate vaccines to Nicꢀspecific B cells and subsequent
production of antiꢀNic antibody from these B cells. Taken together, computational modeling and
competition ELISA approaches offer a simple in vitro preꢀscreening strategy to facilitate the
testing of new HPs with linker modifications that may improve Nic vaccine immunogenicity.
RESULTS
Experimental Rationale. We hypothesized that the accessibility of a given HP to its cognate
Nic specific B cell clone could be assessed by its binding activity to known antiꢀNic antibodies.
To test this hypothesis, we took advantage of both polyclonal and monoclonal antiꢀ1’Nic
antibodies derived from mice immunized with 1’NicꢀKLH conjugates mixed with CpG
oligonucleotide (ODN) adjuvant. We conducted a competition ELISA to estimate interactions of
various HPs with antiꢀNic antibodies. Microplates were coated with HP 1’Nic conjugated to
bovine serum albumin (NicꢀBSA) to capture antiꢀ1’Nic antibody. Various HPs were added to
the antibody prior to their addition to the NicꢀBSA coated wells and the extent to which they
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inhibited antibody binding to the NicꢀBSA was measured. In the absence of inhibitors, the
antibody titers that were predetermined in a linear range served as a control for 100% binding
activity. IC50 value, i.e. the inhibitory concentration of HPs required to inhibit binding of the
antibody to plateꢀcoated NicꢀBSA by 50%, were used to estimate the relative binding affinity,
with lower the IC50 values indicating higher affinity. To make IC50 comparisons across different
microplates, samples, and experiments, free Nic was used as an internal competition control for
each microplate. Thus, ratios of IC50 of HPs to IC50 of free nicotine, i.e., HPꢀ IC50/Nicꢀ IC50,
were used to compare the antibody binding activities of HPs among different experiments, as
well as between different antibodies (e.g., monoclonal and polyclonal antiꢀNic antibodies). From
these comparisons, we can estimate the relative binding affinities of the HPs to the antiꢀ1’Nic
antibodies, inferring their likelihood of interacting with 1’Nicꢀspecific B cells.
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HP Synthesis. In this study, our HP design was focused on the linkers attached to the 1’
pyrrolidyl methyl group. Using 1’Nic HP, that was first reported by Janda’s group, as our
prototype for comparison, we synthesized a series of new Nic HPs (i.e., HP 1ꢀ9), which are
chemically stable and hydrophobic in nature with lengths of 12ꢀ15 Å when fully extended. Given
the previous reports on inferior responses of HPs with some rigid linkers,7 we wanted to use this
feature to test our in vitro screening system, i.e., to investigate whether HPs with rigid linkers
could be selected out by our in vitro assays. In HPs presented here, rigidity of the linker was
increased by incorporating different rings like pyrrolidine in HP 4, piperidine in HP 3 and
piperazine in HP 5 in the linker region, which were away from the Nic core, while other part of
the linker close to the Nic core was kept unchanged. In HPs 1, 2, 5, and 6, we used various ring
systems and reduced alkyl chain length towards the Nic core, while keeping the overall linker
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length closer to the linker used in HP 1’Nic. A cyclohexane ring was introduced in the linker
with trans and cis conformations in HPs 1 and 2 respectively, and a cyclopentane ring was
introduced into the linker of HP 6. An aromatic ring (HP 7) was introduced into the linker to
confer more rigidity than the aliphatic ring of other HPs. Additional rigidity was also achieved
by incorporating unsaturation in the aliphatic chain of HP 8. The position of carbonyl group and
nitrogen atom of an amide functionality was reversed in HP 9 in the linker region and the length
of the linker was longer when compared to other linkers (except 3), as shown in Figure 1. The
synthesis of HP 2 is illustrated in Scheme 1 and described below. The synthetic routes and
characterization of all of the remaining eight Nic HPs (1, 3ꢀ9) are available in the Supporting
Information.
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The syntheses of the nine Nic HPs illustrated in Figure 1 all involved the presence of key
intermediate 16, the synthesis of which is illustrated in Scheme 1. For each of the HPs, a linker
was subsequently added, as exemplified for Nic HP 2 in Scheme 1. The synthesis of Nic HP 2
started from commercially available 5ꢀbromonicotinic acid (Scheme 1), which was converted to
its ethyl ester 11 as a pale yellow solid in 89% yield. Ethyl 5ꢀbromonicotinate (11) underwent
baseꢀmediated condensation with Nꢀvinylpyrrolidinone to afford an intermediate βꢀketoꢀNꢀ
vinyllactam. Acidꢀcatalyzed hydrolysis, decarboxylation and cyclization during basic workup
afforded 5ꢀbromomyosmine (12) as a yellow solid in 70% yield. Imine 12 was then reduced with
sodium borohydride in methanol–acetic acid to afford racemic 5ꢀbromonornic (13) as a pale
yellow liquid in 90% yield. Racemic 13 was resolved7 using αꢀmethoxyꢀαꢀ
(trifluoromethyl)phenylacetic acid (MTPA). Recrystallization gave compounds 14 and 15 as
colorless needles in 54% and 51% yields, respectively. Base washing of (S)ꢀ5ꢀbromonornic (+)ꢀ
MTPA salt (15), followed by catalytic reductive debromination afforded (S)ꢀnornic (16) as a pale
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yellow liquid in 69% yield. Compound 16 was then alkylated with methyl 4ꢀiodobutanoate
affording compound 17 as a pale yellow oil in 78% yield, which upon hydrolysis gave acid
compound 18 as a pale yellow oil (64% yield). Acid 18 was coupled with methyl 4ꢀ
aminocyclohexanecarboxylic acid by using HBTU. This afforded 19 as a pale yellow liquid in
36% yield which, upon hydrolysis, gave the desired Nic HP 2 as a pale yellow liquid in 52%
yield.
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Interactions Between Nic HPs and 1’Nic specific Antibodies. Polyclonal antiꢀ1’Nic
antiserum isolated from mice immunized with 1’NicꢀKLH conjugates mixed with CpG adjuvant
were used to conduct competition ELISA, in which various HPs varying in linker structures were
used as competitors to block the antiserum from binding to plateꢀbound 1’NicꢀBSA. In a
representative competition analysis with pooled antiꢀ1’Nic antiserum, the HPs displayed a wide
range of IC50 binding profiles, some HPs showing much lower IC50 than that of free nicotine
whereas others close to or higher than Nic IC50 (Figure 2). The ratios of HPꢀIC50 to NicꢀIC50 is
summarized in Figure 2B. The ratios closed to or larger than 1.0 mean that the antibody binding
activity of these HPs is comparable to or worse than the binding of free nicotine to the antiserum,
like HPs 1, 2, 6, 7, and 8. On the other hand, the lower IC50 ratios represent the linkers that
confer higher binding activity of HPs to the antiserum, such as HPs 3, 4, and 5, which appeared
to have the binding activity higher than HP 1’Nic that serves as our positive control. These new
HPs, that have linkers with a ring structure, did not seem to interfere with, but slightly increased
their binding activities to the antiꢀ1’Nic antibodies. However, we could not rule out the
possibility that the increased binding activities observed in HPs 3ꢀ5 might have been due to the
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interactions of these HPs with linkerꢀspecific antibodies, which are present in the polyclonal
antiserum and unrelated to Nic specific B cell clones.
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To identify Nicꢀspecific interactions without possible interference from linkerꢀspecific
antibodies, we used a monoclonal antiꢀ1’Nic antibody, mAbꢀ5F3, as a surrogate for a high
affinity 1’Nicꢀspecific B cell clone. This mAb was derived from mice immunized with 1’Nicꢀ
KLH mixed with CpG and was found to have high binding affinity to free Nic with Kd of 66 nM
(Supplemental Information Figure S1A), which was estimated by an equilibrium dialysis assay,
as previously described 15. This binding affinity is comparable to the one of Nicꢀ311, another
wellꢀcharacterized monoclonal antiꢀNic antibody (Supplemental Information Figure S1A). Nicꢀ
311 was derived mice immunized with 3’ꢀaminomethylnicotine conjugated to recombinant
Pseudomonas exoprotein A 16 and had been shown to reduce brain nicotine levels if used as
passive immunization 17. With the availability of mAbꢀ5F3, we wanted to profile its interactions
with our new panel of HPs, thereby getting inference about the HP interactions with the 1’Nicꢀ
specific B cell clone that produces this particular antibody.
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Using the same ELISA competition strategy described above (Figure 2), we examined
HP binding to mAbꢀ5F3. Surprisingly, the binding profile of mAbꢀ5F3 followed a ranking order
(Table 1) similar to the one seen with polyclonal antiꢀ1’Nic antiserum (Figure 2), except HPs 1
and 9. Similar to the finding observed with polyclonal antiserum (Figure 2), HPs 3, 4, and 5 have
relatively higher binding affinities (i.e., low IC50 ratios), and HPs 2, 6, 7, and 8 much lower
affinities than that of HP 1’Nic to the monoꢀspecific antiꢀNic antibody. This finding suggests that
antibody responses after multiple immunizations with 1’NicꢀKLH is either dominated by the
mAbꢀ5F3 like B cell clone, and/or clones closely related to mAbꢀ5F3. To support this argument,
we made sequence comparison between mAbꢀ5F3 and another monoclonal antiꢀ1’Nic antibody
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(NIC9D9), 14, 18 generated by Janda’s group which was also raised specifically to 1’NicꢀKLH.14
We found high homology between these two mAbs at the variable region of the immunoglobulin
heavyꢀ (IgH) and lightꢀ chain (IgL). In particular, both clones possess the same germline VH1 and
Vκ1, similar D2, and somewhat different JH and Jκ gene segments (Supporting Information,
Figure S1B). The high degree of sequence similarities at the variable regions of the two
independent antiꢀ1’Nic mAbs points to a limited repertoire of B cell clones involved in the
1’Nicꢀspecific B cell responses. Meanwhile, the use of mAbꢀ5F3 in our analysis alleviates the
ambiguity of HP binding to linkerꢀspecific antibodies, as well as antibodies targeted to linkerꢀ
protein conjugation sites, which are all present in the polyclonal antiserum raised against Nic
conjugate vaccines.
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Conjugation of Nic HP to Streptavidin (SA). We used streptavidin (SA) as a protein
carrier for these Nic HPs, owing to the following properties: i) wellꢀcharacterized crystal
structure and known residues (i.e., K80, K121, K132, and K134) for Nic conjugation on the
protein surface; ii) relatively small monomeric structure and tetrameric forms to make it feasible
to control and characterize HP conjugation efficiency, and to assess the binding valence and
activity of HPꢀSA conjugates to Nicꢀspecific antibodies; iii) highly immunogenic nature;19ꢀ21 and
iv) ability to link antigen and CpG adjuvant together as a complex for enhanced immunity due to
coꢀdelivery of antigen and CpG adjuvant to the secondary lymphoid tissue for an induction of Tꢀ
cell dependent B cell resposes, e.g., by inducing antibody affinity maturation and production of
memory B cells22ꢀ23. These features help facilitate the design and modification of HPꢀSA
conjugates for enhanced immunogenicity. In our initial analysis, the 1’NicꢀSAꢀCpG vaccine
served as a comparator and elicited good Nicꢀspecific antibody responses at a level comparable
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to the one induced by the same HP linked to KLH, which is a more commonly used carrier
protein (Supporting Information, Figure S2). The conjugation of HPs to SA was made via Nꢀ
hydroxysuccinimide (NHS) esters to lysine resides presumably displayed on the surface of SA.
The average number of HPs conjugated to monomeric SA ranges from 2.5 to 3.7 (Supporting
Information, Figure S3), therefore 10ꢀ15 per SA tetramer, according to the MALDI analysis of
conjugated SA monomer, as summarized in Table 2.
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An ideal Nic vaccine should lead to the production of antibodies primarily targeted to
free Nic. One criterion for generating such a response is an appropriate display of the Nic moiety
on the HPꢀSA conjugates for their access to Nicꢀspecific B cells. To examine the display of HPs
on HPꢀSA conjugates with different linker structures, we applied both a computational modeling
approach to assess the interactions between the HP and its carrier protein, SA, and the
competition ELISA to determine the accessibility of the HPs displayed by HPꢀSA conjugates to
mAbꢀ5F3 and compare to the one of the free HPs to the same antibody (Table 1).
Computational Modeling of Molecular Interactions Between HPs and
Streptavidin. Upon conjugation, the interaction of the HP with neighboring amino acids near
the conjugation site of the carrier protein may impact subsequent interactions with Nicꢀspecific B
cells, possibly accounting for linkerꢀcarrier conjugation effect. Depending on the level of their
interactions, they could either increase or decrease the accessibility of the Nic moiety to Nicꢀ
specific B cell clones. On the other hand, strong interactions may block the access of the Nic
moiety to the B cells, and/or create immunogenic epitopes to induce responses not focused on
Nic. Based on these considerations, we reasoned that conjugates with strong HPꢀSA interactions
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might be associated with poor immunogenicity whereas those with low and moderate
interactions might exert little adverse effect to the quality of antibody resposnes. Given the
availability of structural information of SA and various HPs, we conducted computational
modeling to estimate the interaction strength between HPs and SA to predict possible linkerꢀ
carrier conjugation effect. Specifically, we modeled the conformation of different HPs
conjugated to SA using a rigid docking algorithm in the AutoDock4 program, as illustrated in
Supporting Information Figure S4, and predicted the binding energy of these HPs with SA. The
lowest binding energy was calculated at all four lysine positions for each HP (Table S1). The
sum of the binding energy predicted for HPꢀSA interactions of the conjugates was ranked from
high to low, as presented from left to right, respectively, on Figure 3. The low binding energy
reflects a strong interaction between HP and SA, therefore, exerting high linkerꢀcarrier
conjugation effect. For example, conjugates 1, 2, 6, 7 and 8 display lower binding energy than
the other conjugates, reflecting their possible strong interactions with SA, and therefore, possibly
a high linkerꢀcarrier carrier conjugation effect associated with these conjugates. Interestingly,
among these five HPꢀSAs, four contain HPs (i.e., HPs 2, 6, 7, and 8) that have more rigid linkers
(Figure 1) and they also demonstrate relatively poor binding activities to mAbꢀ5F3 (Table 1 and
Figure 2). Thus, the rigid linkers of these HPs appear not only to block HP accessibilities to
1’Nicꢀspecific mAbꢀ5F3 and polyclonal antiserum, upon conjugation, they might also confer a
strong linkerꢀcarrier carrier conjugation effect, which may compromise the immunogenicity of
these conjugates for generating Nicꢀfocused responses.
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Interactions Between HP-SA Conjugates and Anti-1’Nic mAb. To estimate the
antibody binding activities of the HPs displayed by the HPꢀSA conjugates, we conducted the
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competition ELISA using mAbꢀ5F3 on all the HPꢀSA conjugates to derive their antibody binding
IC50. As compared to the IC50 values of their free HPs, the HPꢀSA conjugates clearly showed
reduced IC50 among all the conjugates, indicating their increased binding activities to the
antibody. The extent of the increases in binding activities was expressed by the ratio of HPꢀIC50
to conjugateꢀIC50, i.e., HPꢀIC50/HPꢀSAꢀIC50 ratios (see the 5th column of Table 1). Two factors
could contribute to the enhanced binding activity, 1) avidity increase due to increased Nic
valences on the conjugates and 2) affinity increase intrinsic to the HPꢀSA conjugates. The
increased avidity of the conjugate should be approximate to the sum of binding affinity of a free
HP to the antibody, or slightly higher, presumably owing to synergized interactions between
multivalent HPs on the conjugates and the biꢀvalent IgG antibody. The increased affinity of the
HP linked to SA may result from structural changes of the HP and/or new epitopes created near
the HPꢀSA conjugation sites, which could reduce an elicitation of Nicꢀfocused responses.
According to the avidity scenario, the increased binding activity of the HPꢀSA conjugates
determined by the HPꢀIC50/HPꢀSAꢀIC50 ratio should match to the HP valence, i.e., the number of
HPs (average 10ꢀ15) linked to one SA tetramer, as estimated by MALDI analysis. Such a match
was indeed found in conjugates 3, 4, and 1’Nic as their HPꢀIC50/HPꢀSAꢀIC50 ratios (i.e., 16ꢀ19)
were not too different from the number of HPs per SA conjugate (see Table 1 and 2). This
finding suggests that the interaction of these conjugates with mAbꢀ5F3 is dominated by HPꢀ
antibody interactions intrinsic to the HPs and the carrier protein has little influences over these
interactions. The binding raitos for conjugates 5 and 9 are somewhat higher. However,
conjugates 1, 2, 6, 7, and 8 exhibited very high HPꢀIC50/HPꢀSAꢀIC50 ratio, ranging from 97 to
662, which is much higher than the estimated valence values and therefore likely resulted from
an increase in the antibody binding affinity of the conjugates. Interestingly, all these conjugates
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showed lower binding energy predicted from the computational modeling (Figure 3), which
implies strong interactions between conjugating HPs and the residues near the conjugation site of
the carrier protein. This agreement is not unexpected since strong HPꢀSA interactions are likely
to alter the display of HPs on the conjugates, dramatically changing their interactions with Nicꢀ
specific antibodies, which, however, may be irrelevant to ultimate binding to free nicotine. Based
on these two lines of analyses, as well as HP binding to antiꢀ1’Nic antibodies, we predicted that
conjugates 3, 4, 1’Nic, and 5 could function as good candidates, since they have high binding
energy and thereby low HPꢀSA interactions predicted by computational modeling (Figure 3), low
and moderate HPꢀIC50/HPꢀSAꢀIC50 ratios (Table 1), as well as low HPꢀIC50/NicꢀIC50 ratios that
reflect high binding affinities of these HPs to the antiꢀ1’Nic antibodies (Figure 2 and Table 1).
On the contrarary, conjugates 2, 6, 7, and 8 were considered as inferior candidates as they were
at the opposite spectrum of the three measurements listed above. To validate this prediction, we
tested these conjugates directly in vivo using Nic pharmacokinetic analysis to determine their
functional efficacy in blocking the Nic entry into the brain.
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Nic Pharmacokinetics Conferred by HP-SA-CpG Vaccines. Upon binding to
biotinylated CpG, the HPꢀSAꢀCpG complexes were used as Nic vaccines as the complexes
render coꢀdelivery of antigen and adjuvant for better initiation of T cell dependent antibody
responses 23. Mice were immunized 3 times with HPꢀSAꢀCpG complexes on days 1, 21 and 42.
Two months after the third immunization, a final injection of HPꢀSA was given to the mice to
further enhance antibody production, in which CpG was not included since adjuvants were found
to reduce maintanence of antibodyꢀproducing longꢀlived plasma cells 24. Ten days later, the mice
received Nic 0.1 mg/kg s.c,, which is approximately equivalent on a mg/kg basis to the Nic
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absorbed from 7 cigarettes by a smoker. Serum and brain Nic levels were measured 4 minutes
after Nic dosing. In this assay, vaccine efficacy was estimated primarily by the level of Nic
present in the brain and compared to the one of the control group. As shown in Figure 4, the
mice immunized with vaccines 1, 3, 4, 5, 6, and 9, as well as 1’Nic, presented significant
reduction in brain Nic levels. The serum Nic levels in these groups were also elevated compared
to the control. However, except group 4 that shows statistically singnifcant increase in the serum
nicotine levels than the control group, the differences observed in the other groups lack statistical
significance, possibly due to high variabilities within each group or reduction of nicotine as a
result of excretion and/or distribution of nicotine to other tissues. Nevertheless, reduction of
nicotine accumulation in the brain is a good indicator in evaluating vaccine efficacy. In contrast,
mice immunized with vaccines 2, 7, and 8 showed little reduction in brain Nic levels (Figure
4A). This finding indicates that these vaccines failed to induce functional antiꢀNic antibody
responses, which is also consistent with our prediction based on the in vitro analyses. In
particular, we found a positive linear correlation between the average HPꢀIC50/NicꢀIC50 ratios
and the average brain Nic concentration in the immunized mice with R2 = 0.51, p = 0.02 (Figure
4C). Taken together, this simple screening strategy helps exclude the inferior HPꢀSA conjugates
(i.e., vaccines 2, 7, and 8), and identify viable candidates.
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Immunogenicity of the HP-SA-CpG Conjugates: Anti-Nic Antibody Titers and
their Nic-binding Affinity (IC50). The levels of antiꢀNic antibodies were measured by ELISA
with HPꢀBSA (HPꢀBSA) conjugates as coating antigens, in which the same HP was used in both
the vaccination and ELISAꢀbased detection. The Nic binding affinity of the antiserum was
estimated by the competition ELISA with free Nic. Variable levels of antiꢀNic antibodies were
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detected in the immunized mice that showed significant changes in brain Nic levels, i.e., those
with vaccines 1, 3, 4, 5, 6, 9, and 1’Nic (Figure 5A). As compared to the group 7 that is a nonꢀ
responder, in term of changing nicotine pharmacokinetics (Figure 4B), groups 1, 3, 4, 5, 6, and 8
showed elevated antibody titers although the antibodies made in group 8 had an extremely high
IC50 value (>50,000 nM), therefore were rather ineffective. Interestingly, the antibody titers
generated from vaccine 1’Nic were quite variable, and therefore, they showed no statistically
significant difference from the antibody titer in group 7 (Figure 4). The lower immunogenicity of
vaccine 1’Nic than the newly designed HPs was observed in several independent experiments;
one representative experiment is shown in Supplemental information, Figure S5.
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At the other extreme, low antibody titers were detected in the mice immunized with
vaccines 2 and 7. This finding is consistent with their Nic pharmacokinetics profiles (Figure 4),
indicating the lack of Nicꢀneutralizing antibodies in these immunized mice. Although the
vaccination group 8 showed high antibody titers, the mice failed to change the Nic distribution
(Figure 4). The extremely high IC50 value (>50,000 nM) of the antiserum in this group explains
the lack of functional efficacy of the generated antibodies, therefore, reflecting the poor quality
of the antiꢀNic antibody response elicited by vaccine 8. In comparing the immunogenicity and
functional activity among all the immunization groups, we did find an inverse correlation
between the average of antibody titers and the average of brain Nic concentration per
immunization group with R2 = 0.55, p = 0.014 (Figure 5C). In addition, the antibody titers of
individual immunized mice were also inversely correlated with the brain concentrations in these
same mice wth R2 = 0.31, p = 10ꢀ7 (Supplemental Information, Figure S6).
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DISCUSSION
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In this study, by applying two in vitro screening strategies, i.e., competition ELISA and
computational molecular modeling, we analyzed a group of newly synthesized Nic HPs with
different linkers attached to the 1’ position of Nic for evaluating their potential as vaccine
candidates. The prediction ranked by the ratios of HP IC50/NicꢀIC50 was supported by the in vivo
Nic pharmacokinetic analysis. In particular, we observed a positive correlation between the
averages of HP IC50/NicꢀIC50 ratios in binding to mAbꢀ5F3, and the reduction of nicotine
concentration in the brain in various immunization groups (R2= 0.51, p =0.02, Figure 4C). The
strength of this correlation is comparable to that of the inverse correlation between the averages
of serum antiꢀNic antibody titers elicited by these vaccines in vaccinated mice and their in vivo
pharmacokinetic efficacy (R2= 0.55, p =0.014, Figure 5C). This finding lends support for the in
vitro antibodyꢀbased profiling as a preꢀimmunization tool in HP selection.
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For HPꢀSA conjugates, givent both positive and negative influences possibly exerted by
linkerꢀcarrier carrier conjugation effect on Nicꢀspecific antibody response, we were not surprised
to find the lack of linear correlations between the total binding energy values of HPꢀSA
interactions calculated from the computational modeling and brain Nic concentrations
(Supplemental Information, Figure S7A), and between their antibody binding ratios of HPꢀ
IC50/HPꢀSAꢀIC50 derived from competition ELISA and brain Nic concentrations (Supplemental
Information, Figure S7B). Nevertheless, these two analyses help exclude HPꢀSA conjugates with
strong linkerꢀcarrier carrier conjugation effect and offer some insight into the contribution of HPꢀ
protein interactions to the immunogenicity of conjugate vaccines. The conjugates 2, 7 and 8 that
were predicted as inferior ones by both modeling (high HPꢀprotein interaction) and competition
ELISA were indeed poor vaccines. The lack of functional efficacy of these vaccines may be due
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to their inability to interact with Nicꢀspecific B cells (e.g., conjugates 2 and 7), and/or their
reactions directed toward nonꢀNic epitopes, like vaccine 8 that induced a high antibody titer with
very low Nicꢀbinding affinity (Figure 5A and B). On the other hand, conjugates that showed low
and moderate degrees of HPꢀSA interactions and exhibited low or moderate antibodyꢀbinding
ratios of HPꢀIC50 to HPꢀSAꢀIC50 might be more viable candidates.
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One exception in our prediction is vaccine 6, which was predicted to be inferior by both
competition ELISA and computational modeling (see Table 1 and Figure 3). This vaccine
actually turned out to be effective in inducing the production of Nicꢀbinding antibodies (Figure
5) and reducing Nic distribution to brain (Figure 4). This finding points out that factors other
than the parameters measured in this study may also influence the immunogenicity of vaccine 6.
Nevertheless, the functional activity of vaccine 6 may be at a borderline since their serum Nic
concentration appeared to be the lowest among all the vaccination groups that demonstrated
functional effect in Nic neutralization (Figure 4A). Therefore, the exclusion of Nicꢀconjugate 6
by our in vitro screenings should not impact the enrichment and selection of good candidates.
For these new newly synthesized HPs, we introduced different degrees of rigidity in the
linker. Among them, HPs 7 and 8 are the most rigid. This rigidity may account for their low
binding affinities to the antiꢀ1’Nic antibodies and strong interactions with carrier protein upon
conjugation (i.e., high IC50, see Tables 1 and Figure 2B). It has been reported by several groups
that HPs with rigid linkers are poor vaccines.7ꢀ8 However, many of our good candidates,
including HPs 1, 3, 4, 5, and 9, display higher rigidity than the control 1’Nic. These vaccines
induced no less or but possibly better (like vaccine 4) responses than the one elicited by vaccine
1’Nic (Figures 4, Figure 5, and Supplemental Information Figure S5). In particular, HPs 3ꢀ5,
possess a linker structure very similar to the one of HP 1’Nic, except an inclusion of different
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ring structures near the conjugation site. This linker modification seems to enhance the binding
activities of these HPs to the antiꢀ1’Nic antibody over HP 1’Nic that was used to raise the
antibody (Table 1). It is possible that the linker with some degree of rigidity can help orient the
Nic epitope for HP interactions with 1’Nicꢀspecific B cells for elicitation of antiꢀ1’Nic antibody
responses. Our screening methods also revealed some subtle differences in the impact of linker
structures. For example, HPs 1 and 2 differed only in isomeric conformation, trans in 1 but cis
in 2. These two conjugates demonstrated different immunogenicity and functionality in vivo
(Figure 4 and 5), which seems correlated with the prediction made by the in vitro screening
strategies (Table 1 and Figure 3).
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In summary we found that an in vitro reverse engineering approach, using either Nicꢀ
specific antiserum or a Nicꢀspecific mAb to screen and rank HPs, correlated well with the in vivo
pharmacokinetic efficacy of immunogens containing these HPs. This approach may allow rapid
in vitro screening of candidate HPs to identify those most likely to produce good immunogens
when conjugated to carriers. A limitation of the study is that it involved only one drug (Nic) and
linkers attached to Nic at just one position. Further study of different HPs and linkers will
determine whether this screening method can be more widely applied to the development of
therapeutic vaccines against other drugs and small chemical compounds.
EXPERIMENTAL PROCEDURES
General Procedures. Reagents and solvents for chemical synthesis were purchased from
Aldrich Chemical Co. or Sigma Chemical Co. and were used without further purification. All
reactions involving airꢀ or moistureꢀsensitive reagents or intermediates were performed under an
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argon atmosphere. Analytical TLC was performed using Silicycle silica gel 60 Å F254 plates
(0.25 mm), and was visualized by UV irradiation (254 nm). Flash chromatography was
performed using Silicycle silica gel (40–60 mesh). Products were concentrated under diminished
pressure. 1H and 13C NMR spectra were obtained using a Varian 400 MHz NMR spectrometer.
Chemical shifts are reported in parts per million (ppm, δ) referenced to the residual 1H of the
solvent (CDCl3, δ 7.26). 13C NMR spectra were referenced to the residual 13C resonance of the
solvent (CDCl3, δ 77.16). Splitting patterns are designated as follows: s, singlet; br s, broad
singlet, d, doublet; br d, broad doublet; dd, doublet of a doublet; t, triplet; q, quartet; m,
multiplet. High resolution mass spectra were obtained at the Arizona State University CLAS
High Resolution Mass Spectrometry Laboratory.
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HP Synthesis. Ethyl 5-Bromonicotinate (11).5 To an iceꢀcooled solution containing 15.0 g
(74.6 mmol) of 5ꢀbromonicotinic acid in 250 mL of ethanol was added dropwise 4 mL of conc
sulfuric acid and the reaction mixture was heated at reflux under argon for 18 h. Ethanol was
removed under diminished pressure and the resulting white residue was dissolved in 100 mL of
water. The aqueous solution was made basic with saturated aq NaHCO3 and extracted with two
150ꢀmL portions of ether. The combined organic extract was washed with 100 mL of brine, dried
over anh MgSO4, and concentrated under diminished pressure to afford 11 as a pale yellow solid:
yield 15.2 g (89%); 1H NMR (CDCl3) δ 1.39 (t, 3H, J = 7.2 Hz), 4.40 (q, 2H, J = 14.8 and 7.6
Hz), 8.40 (t, 1H, J = 2.0 Hz), 8.81 (d, 1H, J = 2.4 Hz) and 9.10 (d, 1H, J = 1.6 Hz); 13C NMR
(CDCl3) δ 14.3, 62.0, 120.7, 127.6, 139.5, 149.0, 154.5 and 164.1.
3-Bromo-5-(4, 5-dihydro-3H-pyrrol-2-yl)pyridine (12). Sodium hydride (3.44 g, 86.0 mmol,
60% dispersion in oil) in a threeꢀnecked flask was washed with three 20ꢀmL portions of hexane.
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The flask was fitted with a reflux condenser, flushed with argon, and charged with 70 mL of
THF. To this was added a solution of 15.2 g (66.1 mmol) of 11 and 7.89 g (71.0 mmol) of 1ꢀ
vinylꢀ2ꢀpyrrolidinone in 15 mL of THF in one portion. The reaction mixture was stirred and
heated at reflux for 60 min and then allowed to cool to room temperature. A solution of 12 mL of
conc HCl in 18 mL of water was added, and the THF was concentrated under diminished
pressure. An additional 18 mL of conc HCl and 36 mL of water were added, and the reaction
mixture was heated at reflux overnight. The reaction mixture was cooled to 0 oC and made basic
with conc aq NaOH and the aqueous phase was then extracted with three 75ꢀmL portions of
CH2Cl2. The combined organic extract was washed sequentially with 100 mL of water and 75
mL of brine, then dried over anh MgSO4 and concentrated under diminished pressure. The
residue was purified by chromatography on a silica gel column (15 × 5 cm). Elution with 19:1
CH2Cl2–acetone afforded 12 as a pale yellow solid: yield 10.3 g (70%); 1H NMR (CDCl3) δ
2.00ꢀ2.08 (m, 2H), 2.86ꢀ2.92 (m, 2H), 4.02ꢀ4.08 (m, 2H), 8.31 (t, 1H, J = 1.6 Hz), 8.67 (d, 1H, J
= 2.0 Hz) and 8.83 (d, 1H, J = 1.6 Hz); 13C NMR (CDCl3) δ 22.6, 34.9, 61.8, 121.0, 131.7,
137.2, 147.1, 152.2 and 169.9.
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3-Bromo-5-(2-pyrrolidinyl)pyridine (13).5 A solution of 2.00 g (8.88 mmol) of 12 in 80 mL
of 4:1 methanol–acetic acid was cooled at –40 oC in a dry ice–acetonitrile bath. To this solution
was added 747 mg (19.8 mmol) of NaBH4 portionwise over a period of 10 min with vigorous
stirring. During the course of the addition, the temperature rose to –20 °C. The reaction mixture
was allowed to warm to ambient temperature, most of the solvent was removed under diminished
pressure, and 200 mL of water was added and made basic with aq NaOH. The aqueous layer was
extracted with three 90ꢀmL portions of CH2Cl2. The combined organic extract was washed with
100 mL of brine, dried over anh K2CO3 and concentrated under diminished pressure. The residue
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was purified by flash chromatography on a silica gel column (10 × 4 cm). Elution with 1:1 ethyl
acetate–methanol afforded racemic 13 as a yellow oil: yield 1.8 g (90%); 1H NMR (CDCl3) δ ꢀ
1.56ꢀ1.66 (m, 1H), 1.81ꢀ1.95 (m, 3H), 2.16ꢀ2.25 (m, 1H), 3.00ꢀ3.06 (m, 1H), 3.11ꢀ3.17 (m, 1H),
4.15 (t, 1H, J = 7.6 Hz), 7.88 (t, 1H, J = 1.6 Hz), 8.46 (d, 1H, J = 2.0 Hz) and 8.50 (d, 1H, J = 2.4
Hz); 13C NMR (CDCl3) δ 25.6, 34.7, 47.1, 59.3, 120.9, 136.8, 142.9, 146.8 and 149.2.
Resolution of Racemic 5-Bromonor Nic. To a stirred solution, 2.33 g (10.3 mmol) of
racemic 13 in 16 mL of ethyl acetate was added a solution of 1.20 g (5.15 mmol) of (–)ꢀαꢀ
methoxyꢀαꢀ(trifluoromethyl)phenylacetic acid [(–)ꢀMTPA] in 4 mL of ethyl acetate. The reaction
mixture was maintained at room temperature for 15 min and the formed crystalline product was
collected by filtration to give (R)ꢀisomer enriched crystals. Three recrystallizations from boiling
acetonitrile yielded 1.28 g (54%) of 14 [(R)ꢀ5ꢀbromonorNic (–)ꢀMTPA salt] as colorless needles.
The filtrate was extracted with two 15ꢀmL portions of 1 N sulfuric acid. The combined
aqueous layer was washed with 20 mL of ether. The aqueous layer was made basic with aq
NaOH, and extracted with three 20ꢀmL portions of CH2Cl2. The combined organic extract was
dried over anh K2CO3 and concentrated under diminished pressure to give an oil enriched in the
(S)ꢀisomer. The oil was dissolved in 9 mL of ethyl acetate and combined with a solution of 1.20
g (5.15 mmol) of (+)ꢀαꢀmethoxyꢀαꢀ(trifluoromethyl)phenylacetic acid [(+)ꢀMTPA] in 4 mL of
ethyl acetate with stirring. The reaction mixture was maintained at room temperature for 15 min,
and the crystallized product was collected by filtration. Three recrystallizations from boiling
acetonitrile yielded 1.20 g (51%) of 15 [(S)ꢀ5ꢀbromonornic (+)ꢀMTPA salt] as colorless needles.
(S)-Nornic (16).5 A suspension of 812 mg (1.76 mmol) of (S)ꢀ5ꢀbromonornic (+)ꢀMTPA salt
in 90 mL of diethyl ether was shaken vigorously with 30 mL of 1 M KOH in a separatory funnel.
The organic layer was washed with 15 mL of 1 M KOH, dried over anh K2CO3 and
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concentrated. The residual oil was dissolved in 30 mL of ethanol and this solution was treated
with 600 µL of Et3N and 100 mg of 10% Pd/C. Hydrogen gas was bubbled through the reaction
mixture for 60 min, then the solution was filtered through a Celite pad and the pad was washed
with 3 mL of ethanol. The filtrate was poured into 90 mL of 1 M aq K2CO3 and extracted with
two 100ꢀmL portions of CH2Cl2. The combined organic extract was washed with 50 mL of brine,
dried over anh K2CO3 and concentrated under diminished pressure. The residue was purified by
flash chromatography on a silica gel column (10 × 1 cm). Elution with 7:1 CH2Cl2–methanol
afforded 16 as a pale yellow oil; yield 179 mg (69%); 1H NMR (CDCl3) δ 1.56ꢀ1.66 (m, 1H),
1.78ꢀ1.91 (m, 2H), 1.99 (br s, 1H), 2.11ꢀ2.20 (m, 1H), 2.96ꢀ3.02 (m, 1H), 3.11ꢀ3.17 (m, 1H),
4.10 (t, 1H, J = 7.6 Hz), 7.17ꢀ7.20 (m, 1H), 7.64ꢀ7.67 (m, 1H), 8.42 (dd, 1H, J = 4.8 and 1.6 Hz)
and 8.54 (d, 1H, J = 2.0 Hz); 13C NMR (CDCl3) δ 25.5, 34.4, 47.0, 60.1, 123.4, 134.1, 140.3,
148.3 and 148.6. This compound had an optical rotation the same as that reported previously.7
(S)-Methyl 4-(2-(pyridin-3-yl)pyrrolidin-1-yl)butanoate (17). A solution of 227 mg (1.21
mmol) of methyl 4ꢀiodobutanoate in 400 µL of acetonitrile was added to a stirred solution of 150
mg (1.01 mmol) of 16 and 0.53 mL (392 mg; 3.03 mmol) of diisopropylethylamine in 800 µL of
acetonitrile at room temperature. After stirring for 18 h, the reaction mixture was concentrated
under diminished pressure and the residue was purified directly by flash chromatography on a
silica gel column (15 × 2 cm). Elution with 5% methanol in CH2Cl2 afforded 17 as a pale yellow
oil: yield 196 mg (78%); silica gel TLC Rf 0.35 (5% methanol in CH2Cl2); 1H NMR (CDCl3) δ
1.55ꢀ1.62 (m, 1H), 1.63ꢀ1.89 (m, 4H), 2.04ꢀ2.19 (m, 4H), 2.25ꢀ2.35 (m, 1H), 2.38ꢀ2.43 (m, 1H),
3.19ꢀ3.30 (m, 2H), 3.52 (s, 3H), 7.18 (dd, 1H, J = 7.6 and 4.8 Hz), 7.62 (d, 1H, J = 8.0 Hz), 8.42
(s, 1H) and 8.47 (s, 1H); 13C NMR (CDCl3) δ 22.7, 23.9, 31.7, 35.2, 51.4, 53.1, 53.2, 67.5, 123.5,
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134.9, 139.6, 148.5, 149.5 and 174.0; mass spectrum (APCI), m/z 249.1606 (M+H)+
(C14H21N2O2 requires m/z 249.1603).
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(S)-4-(2-(Pyridin-3-yl)pyrrolidin-1-yl)butanoic Acid (18). A solution of 63 mg (0.25 mmol)
of 17 in 1 mL of methanol was combined with 30.4 mg (0.76 mmol) of NaOH in 100 ꢀL of H2O
with stirring at room temperature. After 18 h, the reaction mixture was concentrated under
diminished pressure, 2 mL of acetone was added to the residue and the pH was adjusted to 7
using acetic acid. The acetone was concentrated and the crude product was purified directly by
flash chromatography on a silica gel column (10 × 2 cm). Elution with 1:1.5 methanol–CH2Cl2
afforded 18 as a pale yellow oil: yield 38 mg (64%); silica gel TLC Rf 0.3 (1:1.5 methanol–
CH2Cl2); 1H NMR (CDCl3) δ 1.70ꢀ1.80 (m, 3H), 1.82ꢀ2.08 (m, 2H), 2.17ꢀ2.29 (m, 2H), 2.30ꢀ
2.42 (m, 2H), 2.54ꢀ2.61 (m, 1H), 3.44ꢀ3.51 (m, 3H), 7.32 (d, 1H, J = 5.6 Hz), 7.84 (d, 1H, J =
8.0 Hz), 8.50 (s, 1H), 8.55 (s, 1H) and 11.30 (br s, 1H); 13C NMR (CDCl3) δ 22.4, 23.2, 33.8,
34.3, 53.3, 53.6, 67.7, 124.1, 135.9, 137.7, 148.3, 148.9 and 176.6; mass spectrum (APCI), m/z
235.1452 (M+H)+ (C13H19N2O2 requires m/z 235.1447).
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Methyl 4-(4-((S)-2-(pyridin-3-yl)pyrrolidin-1-yl)butanamido)cyclohexanecarboxy-late
(19). To an iceꢀcooled, stirred solution of 38.0 mg (0.16 mmol) of 18 in 700 µL of dry DMF was
added 67.5 mg (178 ꢁmol) of HBTU. After 10 min, a solution of 37.8 mg (195 µmol) of methyl
4ꢀaminocyclohexanecarboxylate hydrochloride and 53.6 µL (49.3 mg; 487 µmol) of Nꢀ
methylmorpholine in 400 µL of dry DMF was added to reaction mixture at 0 oC. The reaction
mixture was allowed to warm to ambient temperature and was then stirred for 18 h. The reaction
mixture was poured into 30 mL of water and extracted with three 20ꢀmL portions of ethyl
acetate. The combined organic extract was washed with 25 mL of brine, dried over anh MgSO4
and concentrated under diminished pressure. The residue was purified by chromatography on a
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silica gel column (15 × 1 cm). Elution with 5% methanol–CH2Cl2 afforded 19 as a pale yellow
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oil: yield (22 mg, 36%); silica gel TLC Rf 0.31 (5% methanol–CH2Cl2); 1H NMR (CDCl3) δ
1.39ꢀ1.51 (m, 2H), 1.61ꢀ1.81 (m, 7H), 1.82ꢀ2.02 (m, 4H), 2.03ꢀ2.10 (m, 1H), 2.14ꢀ2.39 (m, 4H), 2.44ꢀ
2.52 (m, 2H), 3.32ꢀ3.45 (m, 2H), 3.68 (s, 3H), 3.80ꢀ3.90 (m, 1H), 5.85 (br s, 1H), 7.25ꢀ7.31 (m, 1H), 7.69
(d, 1H, J = 6.4 Hz), 8.49 (s, 1H) and 8.55 (s, 1H); 13C NMR (CDCl3) δ 22.5, 24.2, 24.9, 29.0, 34.7, 34.9,
39.7, 46.0, 51.6, 53.4, 53.8, 67.8, 123.5, 135.0, 138.7,148.6, 149.4, 172.2 and 175.4; mass spectrum
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(APCI+), m/z 374.2443 (M+H)+ (C21H32N3O3 requires m/z 374.2444).
4-(4-((S)-2-(Pyridin-3-yl)pyrrolidin-1-yl)butanamido)cyclohexanecarboxylic Acid (2). A
solution of 22.0 mg (59.0 µmol) of 19 in 600 µL of methanol was added 11.8 mg (295 µmol)
NaOH in 100 ꢀL of H2O with stirring at room temperature. After 36 h, the reaction mixture was
concentrated under diminished pressure, 1 mL of acetone added to the residue and the pH was
adjusted to 7 using acetic acid. The acetone was concentrated and the crude product was purified
directly by flash chromatography on a silica gel column (10 × 1 cm). Elution with 2:3 methanol–
CH2Cl2 afforded 2 as a pale yellow oil: yield 11 mg (52%); silica gel TLC Rf 0.25 (2:3
methanol–CH2Cl2); 1H NMR (CDCl3) δ 1.32ꢀ1.39 (m, 1H), 1.42ꢀ1.51 (m, 2H), 1.53ꢀ1.72 (m, 4H),
1.74ꢀ1.88 (m, 3H), 1.91ꢀ2.05 (m, 3H), 2.11ꢀ2.29 (m, 5H), 2.40ꢀ2.49 (m, 2H), 3.32 (t, 1H, J = 8.0 Hz),
3.39ꢀ3.42 (m, 1H), 3.79 (t, 1H, J = 4.4 Hz),6.63 (d, 1H, J = 8.0 Hz), 7.30 (s, 1H), 7.68 (d, 1H, J = 7.6 Hz),
8.46 (s, 1H), 8.75 (s, 1H) and 10.97 (br s, 1H); 13C NMR (CDCl3) δ 22.8, 24.0, 25.7, 29.6, 29.8, 33.9,
35.1, 46.4, 53.6, 53.8, 68.1, 123.8, 136.9, 140.2, 146.8, 148.1, 172.2 and 179.0; mass spectrum
(APCI+), m/z 360.2279 (M+H)+ (C20H30N3O3 requires m/z 360.2287).
Preparation of Free Nic Solution. Nic biꢀtartrate (Sigma) was dissolved in phosphate
buffered saline (PBS) and the absorbance was measured at 260nm using a NanoDrop. The Nic
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extinction coꢀefficient of 2,695 L/(mole·cm) was used to calculate the molar concentration of the
solution. Concentrations are expressed as those of the base.
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HP Conjugation. To ensure consistency of HP synthesis and HP conjugation to the carrier
protein, the effort was made to prepare all HPs within a short period of time (less than 30 days).
Moreover, all the newly synthesized HPs were kept in carboxylic acid form at ꢀ20oC to minimize
degradation. Right before their conjugation, the HPs were converted into active form, i.e.,
succinimidyl ester for conjugation. For conjugation, HPs were dissolved in anhydrous DMSO at
concentrations around 60 mM. Streptavidin (SA, MP Biochemicals, Cat #08623001) and bovine
serum albumin (BSA) were dissolved in 0.1 M sodium bicarbonate buffer, pH 8.3, at a
concentration of 2 mg/mL, and keyhole limpet hemocyanin (KLH, Sigma Cat # H7017) was
reconstituted in PBS at a concentration of 10 mg/mL and diluted in 0.1 M sodium bicarbonate
buffer, pH 8.3, at a final concentration of 2 mg/mL. NHS ester activated Nic HPs were added to
the carrier protein solution, with the molar ratio of HP: carrier protein at 200: 1 for SA, 100:1 for
BSA and 1000: 1 for KLH. The HP/carrier protein mixture was then shaken at room temperature
overnight. Unreacted Nic HP was removed by dialysis in 1X PBS with 6,000 Dalton dialysis
membrane (Spectrum Labs). The HP conjugation ratio was estimated by SA protein mass change
before and after the conjugation reaction (Supporting Information, Figure S3), which was
analyzed on a MALDIꢀTOF instrument in the Proteomics Laboratory of Arizona State
University.
Immunization. Female BALB/c mice were obtained from Charles River Laboratories and
maintained in a pathogenꢀfree animal facility at the Arizona State University Animal Resource
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Center. All mice were handled in accordance with the Animal Welfare Act and Arizona State
University Institutional Animal Care and Use Committee (IACUC). Before experimental
treatment, the mice were randomly distributed in cages and allowed to acclimate for at least 1
week prior to vaccination. Each 6ꢀ8 week old mouse was immunized s.c. with 5 µg (100 pmole)
HPꢀSA incubated with 2 µg (300 pmole) biotinylated CpG ODN 1826 (5’ /5biosg/T*C*C*
A*T*G* A*C*G* T*T*C* C*T*G* A*C*G* T*T 3’, * indicates phosphothioate backbone
modification), or a mixture of 18 µg NicꢀKLH and 2 µg CpG ODN 1826 (5’ T*C*C* A*T*G*
A*C*G* T*T*C* C*T*G* A*C*G* T*T 3’, * indicates phosphothioate backbone modification), at
weeks 0, 3, and 6. Blood was collected at various times from facial veins in accordance with the
Arizona State University and Minneapolis Medical Research Foundation IACUC guidelines and
centrifuged at 2,000 × g for 10 min to collect serum. Two months after the third immunization,
the mice received a final i.p. injection of HPꢀSA (without CpG adjuvant) to further enhance the
antibody levels and 7 days later mouse serum were collected for titer and affinity analysis.
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Nic pharmacokinetics. Ten days after the final challenge with HPꢀSA, mice were injected
s.c. with 0.1 mg/kg Nic (weight of the base) dissolved in PBS. Mice were decapitated 4 min
later, and the brain and blood were collected for Nic concentration analysis by gas
chromatography.8, 12 Brain Nic concentrations were corrected for brain blood content.25
Assessment of Antibody Titers and Relative Affinity. Nunc MaxiSorp 96ꢀwell plates
(Thermo Fisher Scientific) were coated overnight at room temperature with NicꢀBSA using the
same Nic HP as for HPꢀSA (NicꢀBSA, 1 µg/mL). The conjugation and purification of NicꢀBSA
and HPꢀSA were performed in parallel. The antigenꢀcoated plates were washed and blocked with
blocking buffer (1% BSA, 0.1% NaN3, and 0.25% Tweenꢀ20 in PBS) at 37 °C for 1 h and then
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