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

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.     

Nicotine antibody production: comparison of two nicotine conjugates in different animal species.

Preparation and characterization of two nicotine-albumin conjugates, N-Succinyl-6-amino-DL-nicotine-BSA and 6-(Σ-aminocapramido)-DL-nicotine-BSA are described. Ten and eleven molecules of nicotine per molecule of BSA were found, respectively. Two different animal species were used to produce anti-nicotine antibodies. Goats inoculated with 6-(Σ-aminocapramido)-DL-amino nicotine conjugate produced antibodies with higher titer and better affinity and specificity. Comparative studies of antibody production, purification, solid phase assays in controlled pore glass, titration and specificity are also reported.     

An investigation of antibody acyl hydrolysis catalysis using a large set of related haptens

An aspect of catalytic antibody research that receives little attention in the literature involves hapten systems that fail to elicit antibody catalysts despite a high affinity immune response and hapten designs that resemble those known to elicit catalysts. We have investigated a series of 12 phosphate and phosphonate haptens in a total of three animal systems. Dramatic and reproducible differences were observed in the catalytic activities of polyclonal antibodies elicited by the different haptens. A phosphate hapten with a phenyl ring on the side of the hapten opposite the linker elicited reproducibly high levels of polyclonal antibody catalytic activity. The other 11 haptens, most with benzyl groups on the side of the hapten opposite the linker, elicited immune responses in which catalytic activity was significantly weaker in terms of the level of observed catalytic activity, as well as frequency of elicited catalysts. Our results indicate that subtle features of transition state analogue hapten structure can have a dramatic and reproducible influence over the catalytic activity of elicited antibodies in related haptens. Whatever the explanation, subtle changes in mechanistic features due to altered leaving group ability/location or overall hapten flexibility, the comprehensive data presented here indicate that phenyl or 4-nitrophenyl leaving groups located opposite the hapten linker are to be preferred in order to elicit highly active antibody catalysts for acyl hydrolysis reactions.     

A structurally diversified linker enhances the immune response to a small carbohydrate hapten

The tether employed to covalently attach β-mannan disaccharide glycoconjugates influences the specificity of rabbit antibodies that protect against Candida albicans. Two glycoconjugates containing (1?→?2)-β-mannan disaccharides linked to chicken serum albumin (CSA) either via a structurally uniform or via a stereodiversified spacer were prepared and evaluated in immunization trials in mice and rabbits. Immunization with conjugate vaccine possessing a structurally diversified linker induced higher IgG titers against Candida albicans cell wall phosphomannan than a conjugate with a structurally uniform linker. These results suggest that affinity maturation and the specific antibody response can be shifted towards recognition of the desired hapten by employing a linker with diversified configuration.     

Hapten synthesis for enzyme-linked immunoassay of the insecticide triazophos

Two haptens of the insecticide triazophos (O,O-diethyl O-[1-phenyl-1H-1,2,4-triazol-3-yl] phosphorothioate) were synthesized by introducing appropriate spacers in the O-ethyl site of the analyte molecular structure. First, thiophosphoryl chloride (PSCl(3)) reacts with methanol at low temperature to give O-ethyl dichlorothiophosphate. After reacting with 1-phenyl-3H-1,2,4-triazol, the O-ethyl dichlorothiophosphate was transformed into the intermediate O-ethyl O-(1-phenyl-1H-1,2,4-triazol-3-yl) chlorothiophosphate. Then the intermediate reacts with 4-aminobutyric acid and 6-aminobutyric acid to produce hapten I and hapten II, respectively. The molecule structures of the two haptens were identified by (1)H nuclear magnetic resonance spectrum and mass spectrum. An enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody was also developed to evaluate the two haptens. Results showed that the monoclonal antibodies with high titers were obtained after immunizing with protein conjugates of these haptens and that the immunoassay has high affinity and specificity to triazophos. These results suggested that the haptens were synthesized successfully and could be used for immunoassay for the rapid screening and sensitive determination of this insecticide.     

Pushing antibody-based labeling systems to higher sensitivity by linker-assisted affinity enhancement

The sensitivity of antibody/hapten-based labeling systems is limited by the natural affinity ceiling of immunoglobulins. Breaking this limit by antibody engineering is difficult. We thus attempted a different approach and investigated if the so-called bridge effect, a corecognition of the linker present between hapten and carrier protein during antibody generation, can be utilized to improve the affinity of such labeling systems. The well-known haptens 2,4-dinitrophenol (2,4-DNP) and 2,4-dichlorophenoxyacetic acid (2,4-D) were equipped with various linkers, and the resulting affinity change of their cognate antibodies was analyzed by ELISA. Anti-2,4-DNP antibodies exhibited the best affinity to their hapten when it was combined with aminobutanoic acid or aminohexanoic acid. The affinity of anti-2,4-D antibodies could be enhanced even further with longer aliphatic spacers connected to the hapten. The affinity toward aminoundecanoic acid-2,4-D derivatives, for instance, was improved about 100-fold compared to 2,4-D alone and yielded detection limits as low as 100 amoles of analyte. As the effect occurred for all antibodies and haptens tested, it may be sensible to implement the bridge effect in future antibody/hapten-labeling systems in order to achieve the highest sensitivity possible.     

Artificial peptide and carbohydrate antigens. Immobilization of haptens and adjuvant (MDP) on polyacrylamide

To study the influence of the polyacrylamide carrier on immunogenic properties of the peptide and oligosaccharide haptens, we have prepared artificial antigens by conjugation of a synthetic hexapeptide (homologous to the fragment 95-100 of the murine H-2Db antigen heavy chain) or of an oligosaccharide (antigenic determinant of human blood groops, Lea) with polyacrylamide. In some cases the conjugates containing also a synthetic glycopeptide adjuvant, N-acetylmuramoyl-L-alanyl-D-isoglutamine (MDP), were used. Antisera against haptens were obtained by immunization of BALB/c mice with corresponding conjugates. By the enzyme-linked immunosorbent assay it was shown that these antisera had a high binding titer (up to 10 000) to corresponding hapten, and MDP immobilized on the same carrier as hapten possessed a considerable immunostimulating activity. Thus, usefulness of polyacrylamide for preparation of immunogenic artificial molecules carrying peptide and oligosaccharide haptens was demonstrated.     

Monoclonal anti-idiotypic antibodies that recognize the binding site for nicotine on rat brain receptor

Anti-idiotypic monoclonal antibodies have been prepared that represent the internal image of nicotine and are specific for the nicotine binding site on rat brain receptor. Specificity of these antibodies for the combining site on anti-nicotine was demonstrated by their ability to inhibit binding of monoclonal anti-nicotine to immobilized nicotine-polylysine. Furthermore, purified rat brain nicotine receptor but not acetylcholine receptor from fish electric organ effectively competed with anti-nicotine for immobilized nicotine and for immobilized anti-idiotype. Only 9 pmoles of naturally occurring (-)-nicotine inhibited idiotype-anti-idiotype binding by 50% whereas 11 times more (+)-nicotine was required. Acetylcholine, several cholinergic agonists and antagonists, nicotine metabolites, and other structurally related compounds were poor inhibitors.     

The antibody-enzyme analogy. Characterization of antibodies to phosphopyridoxyltyrosine derivatives.

Stable analogs of the crucial Schiff base intermediate of enzymatic and nonenzymatic pyridoxal phosphate catalysis have been used as haptens for induction of specific antibodies. N-(5-phosphopyridoxyl)-3'-amino-L-tyrosine and its conformationally distinct cyclized derivative resemble the Schiff base formed upon mixing tyrosine with pyridoxal phosphate. These compounds were covalently coupled to a protein carrier via the 3'-amino group so as to confer a prescribed orientation, with the coenzyme region farthest removed from the carrier. A third antigen, with the phosphopyridoxyl group alone as the hapten, was prepared by linkage of pyridoxal phosphate directly to free amino groups on the carrier protein. Antibodies elicited for each determinant were purified by means of appropriate affinity columns. Antibody heterogeneity was observed in that different species could be separated from a given serum by sequential elution from the affinity columns with 1 M sodium phosphate buffers of pH 7.6, 5.2, 2.6 and 1.5. In assays of quantitative precipitation, inhibition of precipitation, equilibrium dialysis, and fluorescence quenching, antibodies to the phosphopyridoxyltyrosine haptens showed specificity for the phosphorylated form of the coenzyme and binding activity for both the coenzyme and tyrosine portions of the hapten. Antibodies to the phosphopyridoxyl groups alone did not display a similar reactivity toward the tyrosine portion of the complex haptens. The cyclic and noncyclic conformations of the hapten were serologically distinct, as antibody to each reacted preferentially with the homologous form.     

Active versus passive anti-cytokine antibody therapy against cytokine-associated chronic diseases

Current therapeutic vaccine trials in major chronic diseases including AIDS, cancer, allergy and autoimmunity, target antigenic pathogens but not the pathogenic stromal cytokines which can be major sources of histopathologic processes. Considering that the limited efficacy of these vaccines has been ascribed to local pathogen-induced cytokine dysfunction, we propose to antagonize pathogenic cytokine(s) by high affinity neutralizing auto-Abs triggered by specific anti-cytokine vaccines. As anticipated by theoretical considerations, animal experiments and initial clinical trials showed that anti-cytokine immunization was safe, well tolerated and triggered transient high titers Abs neutralizing pathogenic cytokines but, in contrast to conventional vaccines, no relevant cellular response was observed. Advantages of active versus passive anti-cytokine Ab therapy, particularly for long-term treatments, as those required in AIDS, cancer, allergy and autoimmunity include greater ease of maintaining high Ab titers, lack of anti-antibody responses and low cost.     

High affinity ScFvs from a single rabbit immunized with multiple haptens

We report the generation of single-chain Fv (scFv) fragments with high affinities against four different hapten molecules from a single immunised rabbit. The rabbit was immunised with a mixture of protein conjugates of four different haptens, namely the herbicide mecoprop and derivatives of the herbicides atrazine, simazine, and isoproturon. An scFv phage display library was constructed, and several scFvs with high affinity against each hapten were isolated. For each hapten, a single binder was selected by k(off) ranking and used for affinity determination. The affinities were in sub-nanomolar range and the lowest K(d) value obtained was 6.75 x 10(-10) M. An unusual feature of one of the anti-isoproturon scFvs was its ability to retain binding activity at pH1.7. The utility and potential of using a single animal and immunisation with multiple antigens for the production of multiple, specific, high affinity scFvs by phage display is discussed.     

Immunogenicity of Individual Vaccine Components in a Bivalent Nicotine Vaccine Differ According to Vaccine Formulation and Administration Conditions

Structurally distinct nicotine immunogens can elicit independent antibody responses against nicotine when administered concurrently. Co-administering different nicotine immunogens together as a multivalent vaccine could be a useful way to generate higher antibody levels than with monovalent vaccines alone. The immunogenicity and additivity of monovalent and bivalent nicotine vaccines was studied across a range of immunogen doses, adjuvants, and routes to assess the generality of this approach. Rats were vaccinated with total immunogen doses of 12.5 - 100 μg of 3′-aminomethyl nicotine conjugated to recombinant Pseudomonas exoprotein A (3′-AmNic-rEPA), 6-carboxymethylureido nicotine conjugated to keyhole limpet hemocyanin (6-CMUNic-KLH), or both. Vaccines were administered s.c. in alum or i.p. in Freund’s adjuvant at matched total immunogen doses. When administered s.c. in alum, the contributions of the individual immunogens to total nicotine-specific antibody (NicAb) titers and concentrations were preserved across a range of doses. Antibody affinity for nicotine varied greatly among individuals but was similar for monovalent and bivalent vaccines. However when administered i.p. in Freund’s adjuvant the contributions of the individual immunogens to total NicAb titers and concentrations were compromised at some doses. These results support the possibility of co-administering structurally distinct nicotine immunogens to achieve a more robust immune response than can be obtained with monovalent immunogens alone. Choice of adjuvant was important for the preservation of immunogen component activity.     

Equilibrium and kinetic parameters for the interaction of a monoclonal antibody with liposomes bearing fluorescent haptens

We have obtained equilibrium and rate constants for the interaction of monoclonal IgG and its monovalent Fab fragment with a hapten (fluorescein) attached to the surface of a liposome. Binding was detected at nanomolar hapten concentrations by the quenching of the hapten's fluorescence on antibody binding. The binding parameters were computed from nonlinear least squares fits, using mass-action models. Crypticity of the hapten was observed and interpreted as an equilibrium between two states, extended and sequestered, the latter representing haptens associated with the membrane surface. Depending on the lipid composition of the liposomes, the fraction of sequestered hapten ranged from 0.25 to 0.975; transitions between the two states took place on the time scale of minutes. Fab interactions with extended hapten on the membrane were similar to interactions with water-soluble hapten. The ability of IgG to bind bivalently to membrane gave it an avidity two to six times the affinity for purely monovalent binding. However, the equilibrium constant for the monovalent-bivalent binding equilibrium was effectively four to five orders of magnitude less than that for the initial binding step. This probably reflects steric penalties for the simultaneous binding of two haptens on a membrane.     

Inhibition of functional T cell priming and contact hypersensitivity responses by treatment with anti-secondary lymphoid chemokine antibody during hapten sensitization

Recent studies have suggested a pivotal role for secondary lymphoid chemokine (SLC) in directing dendritic cell trafficking from peripheral to lymphoid tissues. As an extension of these studies, we examined the consequences of anti-SLC Ab treatment during Ag priming on T cell function in an inflammatory response. We used a model of T cell-mediated inflammation, contact hypersensitivity (CHS), where priming of the effector T cells is dependent upon epidermal dendritic cell, Langerhans cells, and migration from the hapten sensitization site in the skin to draining lymph nodes. A single injection of anti-SLC Ab given at the time of sensitization with FITC inhibited Langerhans cell migration into draining lymph nodes for at least 3 days. The CHS response to hapten challenge was inhibited by anti-SLC Ab treatment in a dose-dependent manner. Despite the inhibition of CHS, T cells producing IFN-gamma following in vitro stimulation with anti-CD3 mAb or with hapten-labeled cells were present in the skin-draining lymph nodes of mice treated with anti-SLC Ab during hapten sensitization. These T cells were unable, however, to passively transfer CHS to naive recipients. Animals treated with anti-SLC Ab during hapten sensitization were not tolerant to subsequent sensitization and challenge with the hapten. In addition, anti-SLC Ab did not inhibit CHS responses when given at the time of hapten challenge. These results indicate an important role for SLC during sensitization for CHS and suggest a strategy to circumvent functional T cell priming for inflammatory responses through administration of an Ab inhibiting dendritic cell trafficking.     

Affinity electrophoresis and its applications to studies of immune response

Affinity electrophoresis (AEP) is a useful technique for separation of biomolecules such as plasma proteins, enzymes, nucleic acids, lectins, receptors, and extracellular matrix proteins by specific interactions with their ligands in electric fields and for the determination of dissociation constants for those interactions. Two-dimensional affinity electrophoresis (2-D AEP), which was newly developed by a combination of isoelectric focusing with AEP, has been used for studies on immune response to haptens. Antihapten antibodies, which were induced by immunization of a mouse with the hapten-conjugated bovine serum albumin, were separated by 2-D AEP into a large number of groups of IgG spots with a few microliters of antiserum. Each group of spots showed an identical affinity for the hapten but different isoelectric points as in the case of monoclonal antibodies specific to the hapten. This enabled us to study the diversification and affinity maturation of antihapten antibodies in the course of immunization of a single mouse. Furthermore, effects of a carrier and a hapten array on the production of antihapten antibodies and the cause of charge heterogeneity of monoclonal antibodies were also examined to understand the molecular basis of the immune response in vivo.     

Idiotype-specific T suppressor factor alternatively interacts with a nonspecific T-acceptor-like cell to mediate immune suppression

The ability of the idiotype (Id)-specific second-order T suppressor factor (TsF2) to interact with a final effector Ts cell type other than the previously reported third-order Ts (Ts3) subset was studied in the phenyltrimethylamino (TMA) hapten system. Hence, mice were primed with unrelated heterologous haptens to induce the nonspecific T acceptor (Tacc) cells following published procedures. When enriched T cell populations containing these nonspecific Ts were briefly incubated in vitro with TMA-TsF2, they produced suppression upon adoptive transfer into cyclophosphamide-treated mice which had been previously immunized for TMA-specific delayed-type hypersensitivity. Despite the fact that the effector population studied in this report also required Id-binding TsF2 for its function, it differs markedly from the Ts3 subset studied previously in the TMA system. First, the cell type studied herein could be easily generated with noncrossreacting heterologous chemically reactive haptens when applied directly to the skin of mice. Furthermore, these Ts effector cells had no detectable intrinsic receptors for homologous haptens and most importantly, unlike Ts3, this population had no affinity for the TMA hapten. Nevertheless, the nonspecifically induced Ts once activated by TsF2 suppresses TMA-directed, but not similar immune responses specific for heterologous haptens. Thus the results indicate that TsF2 can functionally interact with a final effector Ts subset (very similar to the Tacc) other than the well described Ts3 population. The ramifications of these findings are discussed with reference to a generalized view of the cellular basis of terminal phases of immune suppression.     

A morphine/heroin vaccine with new hapten design attenuates behavioral effects in rats

Heroin use has seriously threatened public heath in many countries, but the existing therapies continue to have many limitations. Recently, immunotherapy has shown efficacy in some clinical studies, including vaccines against nicotine and cocaine, but no opioid vaccines have been introduced in clinical studies. The development of a novel opioid antigen designed specifically for the prevention of heroin addiction is necessary. A morphine-keyhole limpet hemocyanin conjugate was prepared and administered subcutaneously in rats. Antibody titers in plasma were measured using an enzyme-linked immunosorbent assay (ELISA). Competitive ELISA was used to assess the selectivity of the antibodies. Dopamine concentrations in the nucleus accumbens in rats after vaccine administration were determined by high-performance liquid chromatography with electrochemical detection. The effects of the vaccine on the heroin-primed restatement of self-administration and locomotor sensitization were evaluated. A novel hapten, 6-glutarylmorphine, was produced, and the vaccine generated a high antibody titer response. This vaccine displayed specificity for both morphine and heroin, but the anti-morphine antibodies could not recognize dissimilar therapeutic opioid compounds, such as buprenorphine, methadone, naloxone, naltrexone, codeine, and nalorphine. The morphine antibody significantly decreased morphine-induced locomotor activity in rats after immunization. Importantly, rats immunized with this vaccine did not exhibit heroin-primed reinstatement of heroin seeking when antibody levels were sufficiently high. The vaccine reduced dopamine levels in the nucleus accumbens after morphine administration, which is consistent with its behavioral effects. These results suggest that immunization with a novel vaccine is an effective means of inducing a morphine-specific antibody response that is able to attenuate the behavioral and psychoactive effects of heroin.     

Design of acridinium-9-carboxamides and anti-acridinium antibodies for chemiluminescent signal enhancement

A novel system of signal enhancement is presented in which every labeled antibody is capable of generating a signal. Three chemiluminescent acridinium-9-carboxamide haptens (1, 2, and 3) which incorporated differences in charge and location of the linker were designed and synthesized. Anti-acridinium polyclonal antibodies for each hapten were screened using surface plasmon resonance instrumentation to determine specificity for each hapten. Anti-acridinium 2 antibodies were found to be non-cross-reactive to acridinium 1. This property was exploited to design secondary antibody conjugates which would bind to primary antibodies labeled with 2 yet could still be labeled with the structurally similar acridinium 1. Consequently, both layers contributed to the overall chemiluminescent signal. This format is an advance over other signal amplification formats which employ non-signal-generating, labeled antibodies to construct multilayered systems.     

Thermodynamics of antibody-antigen reactions. 1. The binding of simple haptens to two classes of antibodies fractionated according to affinity

The thermodynamic parameters which characterize the binding of dinitrophenylglycine and dinitrophenylmethoxypoly(ethylene glycol) to selected affinity classes of equine IgG and IgG(T) antibodies were determined by fluorescence quenching and flow calorimetry. The binding enthalpies and entropies were in all cases large and negative, falling in the ranges -14 to -17 kcal/mol and -18 to -25 eu, respectively. The differences in the enthalpies and entropies of binding for different affinity classes and for different haptens are discussed with reference to differences in the structures of the haptens studied and as indications of differences in binding site structure. In addition, the apparent existence of fluorescent side chains which can transfer energy to either hapten binding site in IgG(T) antibodies but not in IgG antibodies is interpreted as indicative of a smaller average interbinding site distance in IgG(T) than in IgG antibodies.