Acute injection of METH reduces ICSS thresholds.

<p>ICSS thresholds (A) and response latencies (B) (expressed as percent of baseline, mean ± SEM) following s.c. injection of METH (0–0.56 mg/kg) in Experiment 1a. ** Significantly different from 0 mg/kg METH, p < 0.01.</p

MAb7F9 modestly attenuates the ability of acute METH to reverse elevations in ICSS thresholds during METH withdrawal.

<p>ICSS thresholds (A) and response latencies (B) (expressed as percent of baseline, mean ± SEM) in rats administered i.v. PBS or 200 mg/kg mAb immediately following cessation of a chronic METH infusion, followed 20 hr later by s.c. SAL or 0.3 mg/kg METH in Experiment 3. *^, ** Significantly different from SAL-injected animals for that mAb dose, p < 0.05 or 0.01. ^#,mAb 200 + METH significantly different from PBS + METH, p < 0.05.</p

Simple Radiometric Method for Accurately Quantitating Epitope Densities of Hapten–Protein Conjugates with Sulfhydryl Linkages

Control of small molecule hapten epitope densities on antigenic carrier proteins is essential for development and testing of optimal conditions for vaccines. Yet, accurate determination of epitope density can be extremely difficult to accomplish, especially with the use of small haptens, large molecular weight carrier proteins, and limited amounts of protein. Here we report a simple radiometric method that uses ¹⁴C-labeled cystine to measure hapten epitope densities during sulfhydryl conjugation of haptens to maleimide activated carrier proteins. The method was developed using a (+)-methamphetamine (METH)-like hapten with a sulfhydryl terminus, and two prototype maleimide activated carrier proteins, bovine serum albumin (BSA) and immunocyanin monomers of keyhole limpet hemocyanin. The method was validated by immunochemical analysis of the hapten–BSA conjugates, and least-squares linear regression analysis of epitope density values determined by the new radiometric method versus values determined by matrix-assisted laser desorption/ionization mass spectrometry. Results showed that radiometric epitope density values correlated extremely well with the mass spectrometrically derived values (<i>r</i>² = 0.98, <i>y</i> = 0.98<i>x</i> + 0.91). This convenient and simple method could be useful during several stages of vaccine development including the optimization and monitoring of conditions for hapten–protein conjugations, and choosing the most effective epitope densities for conjugate vaccines

Combining Active Immunization with Monoclonal Antibody Therapy To Facilitate Early Initiation of a Long-Acting Anti-Methamphetamine Antibody Response

We hypothesized that an anti-METH mAb could be used in combination with a METH-conjugate vaccine (MCV) to safely improve the overall quality and magnitude of the anti-METH immune response. The benefits would include immediate onset of action (from the mAb), timely increases in the immune responses (from the combined therapy) and duration of antibody response that could last for months (from the MCV). A novel METH-like hapten (METH-SSOO9) was synthesized and then conjugated to immunocyanin monomers of keyhole limpet hemocyanin (IC_KLH) to create the MCV IC_KLH-SOO9. The vaccine, in combination with previously discovered anti-METH mAb7F9, was then tested in rats for safety and potential efficacy. The combination antibody therapy allowed safe achievement of an early high anti-METH antibody response, which persisted throughout the study. Indeed, even after 4 months the METH vaccine antibodies still had the capacity to significantly reduce METH brain concentrations resulting from a 0.56 mg/kg METH dose

Development and testing of AAV-delivered single-chain variable fragments for the treatment of methamphetamine abuse

<div><p>Methamphetamine (METH) substance abuse disorders have major impact on society, yet no medications have proven successful at preventing METH relapse or cravings. Anti-METH monoclonal antibodies can reduce METH brain concentrations; however, this therapy has limitations, including the need for repeated dosing throughout the course of addiction recovery. An adeno-associated viral (AAV)-delivered DNA sequence for a single-chain variable fragment could offer long-term, continuous expression of anti-METH antibody fragments. For these studies, we injected mice via tail vein with 1 x 10¹² vector genomes of two AAV8 scFv constructs and measured long-term expression of the antibody fragments. Mice expressed each scFv for at least 212 days, achieving micromolar scFv concentrations in serum. In separate experiments 21 days and 50 days after injecting mice with AAV-scFvs mice were challenged with METH <i>in vivo</i>. The circulating scFvs were capable of decreasing brain METH concentrations by up to 60% and sequestering METH in serum for 2 to 3 hrs. These results suggest that AAV-delivered scFv could be a promising therapy to treat methamphetamine abuse.</p></div

A comparison of METH or AMP brain and serum concentrations over time, after a 0.56 mg/kg <i>ip</i> injection of METH, between AAV-scFv6H4, AAV-scFv7F9, and a saline control at day 21 post AAV8 administration.

<p>Mice treated with either AAV-scFv6H4 or AAV-scFv7F9 showed significantly lower brain METH concentrations (a) and significantly higher serum concentrations of METH (b) than the saline-treated mice (*, p < 0.05; #, p < 0.001). There was also a significant decrease in AMP brain concentrations (c) in the AAV-scFv treated groups compared to control mice but no difference in serum AMP concentrations (d). Points are shown as mean ± SEM (n = 3–4 per group).</p

Comparison of METH and scFv molar concentrations over time.

<p>On day 0, mice were injected with PBS (control treatment), AAV-scFv6H4, or AAV-scFv7F9. Serum scFv6H4 and 7F9 concentrations were measured via ELISA on days 8 and 15. On day 22, mice were treated with METH as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200060#pone.0200060.g006" target="_blank">Fig 6</a>. Both serum scFv and METH concentrations were measured. Mice treated with either AAV-scFv6H4 or AAV-scFv7F9 showed significantly higher serum concentrations of METH than the control mice (*, p < 0.05; #, p < 0.001). Data points are mean ± SEM (n = 3–4 per group).</p

Comparison of IC₅₀ values for METH between culture-produced scFv and expressed AAV-scFv.

<p>A competitive binding assay was performed with each of the variants. The IC₅₀ (in nM) for both scFv and AAV-scFv variants was estimated at 50% ³H-METH bound (dotted line). Individual data points are shown as the mean ± SEM (n = 8 per group).</p