44 research outputs found
Characterization and optimization of heroin hapten-BSA conjugates: method development for the synthesis of reproducible hapten-based vaccines
A potential new treatment for drug addiction is immunization with vaccines that induce antibodies that can abrogate the addictive effects of the drug of abuse. One of the challenges in the development of a vaccine against drugs of abuse is the availability of an optimum procedure that gives reproducible and high yielding hapten-protein conjugates. In this study, a heroin/morphine surrogate hapten (MorHap) was coupled to bovine serum albumin (BSA) using maleimide-thiol chemistry. MorHap-BSA conjugates with 3, 5, 10, 15, 22, 28, and 34 haptens were obtained using different linker and hapten ratios. Using this optimized procedure, MorHap-BSA conjugates were synthesized with highly reproducible results and in high yields. The number of haptens attached to BSA was compared by 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay, modified Ellmanβs test and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Among the three methods, MALDI-TOF MS discriminated subtle differences in hapten density. The effect of hapten density on enzyme-linked immunosorbent assay (ELISA) performance was evaluated with seven MorHap-BSA conjugates of varying hapten densities, which were used as coating antigens. The highest antibody binding was obtained with MorHap-BSA conjugates containing 3β5 haptens. This is the first report that rigorously analyzes, optimizes and characterizes the conjugation of haptens to proteins that can be used for vaccines against drugs of abuse. The effect of hapten density on the ELISA detection of antibodies against haptens demonstrates the importance of careful characterization of the hapten density by the analytical techniques described. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00216-014-8035-x) contains supplementary material, which is available to authorized users
Plasmodium vivax Tryptophan-Rich Antigen PvTRAg33.5 Contains Alpha Helical Structure and Multidomain Architecture
Tryptophan-rich proteins from several malarial parasites have been identified where they play an important role in host-parasite interaction. Structural characterization of these proteins is needed to develop them as therapeutic targets. Here, we describe a novel Plasmodium vivax tryptophan-rich protein named PvTRAg33.5. It is expressed by blood stage(s) of the parasite and its gene contains two exons. The exon 1 encodes for a 23 amino acids long putative signal peptide which is likely to be cleaved off whereas the exon 2 encodes for the mature protein of 252 amino acids. The mature protein contains B-cell epitopes which were recognized by the human immune system during P.vivax infection. The PvTRAg33.5 contains 24 (9.5%) tryptophan residues and six motifs whose patterns were similar among tryptophan-rich proteins. The modeled structure of the PvTRAg33.5 consists of a multidomain architecture which is stabilized by the presence of large number of tryptophan residues. The recombinant PvTRAg33.5 showed predominantly Ξ± helical structure and alpha helix to beta sheet transition at pH below 4.5. Protein acquires an irreversible non-native state at temperature more than 50Β°C at neutral pH. Its secondary and tertiary structures remain stable in the presence of 35% alcohol but these structures are destabilized at higher alcohol concentrations due to the disturbance of hydrophobic interactions between tryptophanyl residues. These structural changes in the protein might occur during its translocation to interact with other proteins at its final destination for biological function such as erythrocyte invasion
A Stable Heroin Analogue That Can Serve as a Vaccine Hapten to Induce Antibodies That Block the Effects of Heroin and Its Metabolites in Rodents and That Cross-React Immunologically with Related Drugs of Abuse
An
improved synthesis of a haptenic heroin surrogate <b>1</b> (6-AmHap)
is reported. The intermediate needed for the preparation
of <b>1</b> was described in the route in the synthesis of <b>2</b> (DiAmHap). A scalable procedure was developed to install
the C-3 amido group. Using the Boc protectng group in <b>18</b> allowed preparation of <b>1</b> in an overall yield of 53%
from <b>4</b> and eliminated the necessity of preparing the
diamide <b>13</b>. Hapten <b>1</b> was conjugated to tetanus
toxoid and mixed with liposomes containing monophosphoryl lipid A
as an adjuvant. The <b>1</b> vaccine induced high anti-<b>1</b> IgG levels that reduced heroin-induced antinociception and
locomotive behavioral changes following repeated subcutaneous and
intravenous heroin challenges in mice and rats. Vaccinated mice had
reduced heroin-induced hyperlocomotion following a 50 mg/kg heroin
challenge. The <b>1</b> vaccine-induced antibodies bound to
heroin and other abused opioids, including hydrocodone, oxycodone,
hydromorphone, oxymorphone, and codeine
Efficacy, but Not Antibody Titer or Affinity, of a Heroin Hapten Conjugate Vaccine Correlates with Increasing Hapten Densities on Tetanus Toxoid, but Not on CRM<sub>197</sub> Carriers
Vaccines against drugs of abuse have
induced antibodies in animals
that blocked the biological effects of the drug by sequestering the
drug in the blood and preventing it from crossing the blood-brain
barrier. Drugs of abuse are too small to induce antibodies and, therefore,
require conjugation of drug hapten analogs to a carrier protein. The
efficacy of these conjugate vaccines depends on several factors including
hapten design, coupling strategy, hapten density, carrier protein
selection, and vaccine adjuvant. Previously, we have shown that <b>1</b> (MorHap), a heroin/morphine hapten, conjugated to tetanus
toxoid (TT) and mixed with liposomes containing monophosphoryl lipid
A [LΒ(MPLA)] as adjuvant, partially blocked the antinociceptive effects
of heroin in mice. Herein, we extended those findings, demonstrating
greatly improved vaccine induced antinociceptive effects up to 3%
mean maximal potential effect (%MPE). This was obtained by evaluating
the effects of vaccine efficacy of hapten <b>1</b> vaccine conjugates
with varying hapten densities using two different commonly used carrier
proteins, TT and cross-reactive material 197 (CRM<sub>197</sub>).
Immunization of mice with these conjugates mixed with LΒ(MPLA) induced
very high anti-<b>1</b> IgG peak levels of 400β1500 ΞΌg/mL
that bound to both heroin and its metabolites, 6-acetylmorphine and
morphine. Except for the lowest hapten density for each carrier, the
antibody titers and affinity were independent of hapten density. The
TT carrier based vaccines induced long-lived inhibition of heroin-induced
antinociception that correlated with increasing hapten density. The
best formulation contained TT with the highest hapten density of β₯30
haptens/TT molecule and induced %MPE of approximately 3% after heroin
challenge. In contrast, the best formulation using CRM<sub>197</sub> was with intermediate <b>1</b> densities (10β15 haptens/CRM<sub>197</sub> molecule), but the %MPE was approximately 13%. In addition,
the chemical synthesis of <b>1</b>, the optimization of the
conjugation method, and the methods for the accurate quantification
of hapten density are described