Novel cocaine vaccine linked to a disrupted adenovirus gene transfer vector blocks cocaine psychostimulant and reinforcing effects

Abstract

Immunotherapy is a promising treatment for drug addiction. However, insufficient immune responses to vaccines in most subjects pose a challenge. In this study, we tested the efficacy of a new cocaine vaccine (dAd5GNE) in antagonizing cocaine addiction-related behaviors in rats. This vaccine used a disrupted serotype 5 adenovirus (Ad) gene transfer vector coupled to a third-generation cocaine hapten, termed GNE (6-(2R,3S)-3-(benzoyloxy)-8-methyl-8-azabicyclo [3.2.1] octane-2-carboxamido-hexanoic acid). Three groups of rats were immunized with dAd5GNE. One group was injected with (3)H-cocaine, and radioactivity in the blood and brain was determined. A second group was tested for cocaine-induced locomotor sensitization. A third group was examined for cocaine self-administration, extinction, and reinstatement of responding for cocaine. Antibody titers were determined at various time-points. In each experiment, we added a control group that was immunized with dAd5 without a hapten. The vaccination with dAd5GNE produced long-lasting high titers (>10(5)) of anti-cocaine antibodies in all of the rats. The vaccination inhibited cocaine-induced hyperlocomotor activity and sensitization. Vaccinated rats acquired cocaine self-administration, but they showed less motivation to self-administer cocaine under a progressive-ratio schedule than control rats. When cocaine was not available in a session, control rats exhibited 'extinction burst' responding, whereas vaccinated rats did not. Moreover, when primed with cocaine, vaccinated rats did not reinstate responding, suggesting a blockade of cocaine-seeking behavior. These data strongly suggest that our dAd5GNE vector-based vaccine may be effective in treating cocaine abuse and addiction.

Figure 1

Timeline of cocaine ambulatory behavior studies. Day 1 (D1) through D72 for the vaccination and cocaine challenges that preceded the ambulatory behavior assays.

Figure 2

Brain and blood partition of cocaine in naive and dAd5GNE (vaccine that used a disrupted serotype 5 adenovirus (Ad) gene transfer vector coupled to a third-generation cocaine hapten, termed GNE (6-(2R,3S)-3-(benzoyloxy)-8-methyl-8-azabicyclo [3.2.1] octane-2-carboxamido-hexanoic acid))-vaccinated rats. (a) Levels of cocaine in the brain (ng/g brain). (b) Blood levels of cocaine (ng/ml serum) in naive and dAd5GNE-vaccinated rats challenged with cocaine. Cocaine assays were conducted at 2 min following the intravenous administration of cocaine (25.0 μg containing 3.0 μCi ³H-cocaine) to rats 6 weeks after the fourth immunization with dAd5GNE (n=5). Comparisons between groups were conducted by one-way paired two-sample t-test.

Figure 3

Persistence of inhibition of cocaine-induced hyperlocomotor activity in dAd5GNE (vaccine that used a disrupted serotype 5 adenovirus (Ad) gene transfer vector coupled to a third-generation cocaine hapten, termed GNE (6-(2R,3S)-3-(benzoyloxy)-8-methyl-8-azabicyclo [3.2.1] octane-2-carboxamido-hexanoic acid))-vaccinated rats. Comparison of naive and dAd5GNE-immunized rats assessed by cocaine-induced locomotor activity upon repeated exposures over an 18-day period with eight cocaine challenges. Vaccinated and naive+cocaine rats were sensitized to cocaine (15 mg/kg, i.p.) during days 1–4 and then challenged biweekly with cocaine (15 mg/kg, i.p.) during days 7–18 (n=8 per group). (a) The total distance traveled inside an infrared beam-monitored open-field apparatus (AccuScan, 40 × 40 cm²) for 30 min immediately following cocaine or phosphate-buffered saline (PBS) challenge was assessed for each trial. Rats from each experimental group were initially injected with PBS to establish baseline activity (data points at day 0). Comparisons between test groups used a non-parametric one-way analysis of variance (ANOVA) Kruskal–Wallis test: dAd5GNE+cocaine (▪) vs naive+cocaine (•), H=9.1, p<0.01; dAd5GNE+cocaine vs naive+PBS (Δ), H=3.4, p>0.05; naive+cocaine vs naive+PBS, H=11.3, p<0.001. Repeated-measures ANOVA was performed on the biweekly challenges: dAd5GNE+cocaine vs naive+cocaine, F=5.6, p<0.05; dAd5GNE+cocaine vs naive+PBS, F=12.4, p<0.01; naive+cocaine vs naive+PBS, F=22.8, p<0.001. The repeated-measures ANOVA within groups revealed no significant differences among the repetitions (dAdGNE, F=0.8, p>0.5; naive+cocaine, F=0.2, p>0.5; naive+PBS, F=2.8, p>0.05). (b) Total vertical activity time for 30 min immediately following PBS or cocaine (15 mg/kg) challenge was plotted for each trial (n=8 per group). The amount of time spent displaying vertical activity (ie, breaking z axis beams) is plotted for each challenge event. Kruskal–Wallis test comparisons: dAd5GNE+cocaine (▪) vs naive+cocaine (•), H=4.8, p<0.05; dAd5GNE+cocaine vs naive+PBS (Δ), H=0.5, p>0.1; naive+cocaine vs naive+PBS, H=5.8, p<0.05. Repeated-measures ANOVA was performed on the biweekly challenges: dAd5GNE+cocaine vs naive+cocaine, F=9.6, p<0.01; dAd5GNE+cocaine vs naive+PBS, F=0.2, p>0.5; naive+cocaine vs naive+PBS, F=13.3, p<0.01. Repeated-measures ANOVA within groups for cocaine challenge revealed no significant differences (F=0.1, p>0.5).

Figure 4

Timeline of cocaine self-administration. Day 1 (D1) through D102 for the immunization, catheterization, and self-administration studies.

Figure 5

dAd5GNE (vaccine that used a disrupted serotype 5 adenovirus (Ad) gene transfer vector coupled to a third-generation cocaine hapten, termed GNE (6-(2R,3S)-3-(benzoyloxy)-8-methyl-8-azabicyclo [3.2.1] octane-2-carboxamido-hexanoic acid)) evoked anti-cocaine antibodies in rats. Total anti-cocaine immunoglobulin G (IgG) antibody titers over time. Male Wistar rats were vaccinated intramuscularly with 10 μg dAd5GNE formulated in Aduplex or non-conjugated disrupted Ad5 (negative control). Rats (n=12) were administered vaccine at 0, 3, 5, and 10 weeks, and the antibody titers were assessed by enzyme-linked immunosorbent assay (ELISA) at 0, 2, 4, 7, 9, 12, 20, and 22 weeks. The data represent the animals used in the self-administration model.

Figure 6

Effect of dAd5GNE (vaccine that used a disrupted serotype 5 adenovirus (Ad) gene transfer vector coupled to a third-generation cocaine hapten, termed GNE (6-(2R,3S)-3-(benzoyloxy)-8-methyl-8-azabicyclo [3.2.1] octane-2-carboxamido-hexanoic acid)) on cocaine self-administration. Rats were immunized with dAd5GNE (10 μg per rat) and catheterized with intravenous catheters 1 week after the last boost. After recovery, they were allowed to self-administer 0.5 mg/kg per injection of cocaine in 1 h sessions under a fixed-ratio (FR) schedule. After 20 sessions, the cocaine dose–response function and responding for cocaine were determined under FR and progressive-ratio (PR) schedules, respectively. As a control, rats that were treated with dAd5 without a hapten underwent the same procedure as the vaccine group. At the end of the study, the rats were tested for food self-administration under a PR schedule. (a) Acquisition of cocaine self-administration under an FR schedule. The data are expressed as the number of cocaine injections on the left and cocaine intake (mg/kg) on the right. (b) Cocaine dose–response function. The data are expressed as the number of injections per hour at each dose of cocaine. The lines are linear regression lines. ^**p<0.01, ^***p<0.001, compared with responding for 0.5 mg/kg per injection of cocaine. (c) Cocaine self-administration under a PR schedule. The data are expressed as the number of injections per session on the left and ratio requirement per injection on the right. ^**p<0.01, between groups.

Figure 7

Extinction and reinstatement of responding for cocaine. During the extinction session, cocaine was replaced with saline, and responding on the previously cocaine-associated lever resulted in saline delivery without the cue light. For the reinstatement test, the rats were intraperitoneally injected with saline or cocaine (10 or 15 mg/kg) and immediately placed in an operant chamber. During the reinstatement session, responding on the previously cocaine-associated lever resulted in saline delivery with the cue light. (a) Extinction. ^*p<0.05, ^**p<0.01, ^***p<0.01, compared with extinction session 1; ^##p<0.01, compared with dAd5GNE group in the same session. (b) Reinstatement. ^**p<0.01, compared with saline.