Effect of currently approved carriers and adjuvants on the pre-clinical efficacy of a conjugate vaccine against oxycodone in mice and rats

Abstract

Vaccination against the highly abused prescription opioid oxycodone has shown pre-clinical efficacy for blocking oxycodone effects. The current study further evaluated a candidate vaccine composed of oxycodone derivatized at the C6 position (6OXY) conjugated to the native keyhole limpet hemocyanin (nKLH) carrier protein. To provide an oxycodone vaccine formulation suitable for human studies, we studied the effect of alternative carriers and adjuvants on the generation of oxycodone-specific serum antibody and B cell responses, and the effect of immunization on oxycodone distribution and oxycodone-induced antinociception in mice and rats. 6OXY conjugated to tetanus toxoid (TT) or a GMP grade KLH dimer (dKLH) was as effective as 6OXY conjugated to the nKLH decamer in mice and rats, while the 6OXY hapten conjugated to a TT-derived peptide was not effective in preventing oxycodone-induced antinociception in mice. Immunization with 6OXY-TT s.c. absorbed on alum adjuvant provided similar protection to 6OXY-TT administered i.p. with Freund's adjuvant in rats. The toll-like receptor 4 (TLR4) agonist monophosphoryl lipid A (MPLA) adjuvant, alone or in combination with alum, offered no advantage over alum alone for generating oxycodone-specific serum antibodies or 6OXY-specific antibody secreting B cells in mice vaccinated with 6OXY-nKLH or 6OXY-TT. The immunogenicity of oxycodone vaccines may be modulated by TLR4 signaling since responses to 6OXY-nKLH in alum were decreased in TLR4-deficient mice. These data suggest that TT, nKLH and dKLH carriers provide consistent 6OXY conjugate vaccine immunogenicity across species, strains and via different routes of administration, while adjuvant formulations may need to be tailored to individual immunogens or patient populations.

Figure 1. Drug and immunogen structures.

Oxycodone and hydrocodone free drugs, and representation of hapten conjugated to carriers. The lead 6OXY hapten was conjugated to native KLH decamer (nKLH), GMP grade KLH dimer (dKLH), tetanus toxoid (TT) and a TT-derived peptide.

Figure 2. The ability of vaccination with different carrier-containing immunogens in preventing oxycodone nociception and its distribution to the brain in BALB/c mice.

A) Oxycodone antinociception in mice immunized with either 6OXY-TT, 6OXY-TT peptide or unconjugated TT. The % maximal possible effect (MPE%) is calculated as (post-drug – baseline)/ (maximal cutoff-baseline) * 100; and B) Oxycodone distribution to the brain in the same mice as in previous panel. In both panels, percent (%) decrease compared to TT control is shown on top of each group. Statistical symbols: * p< 0.05 compared to TT control.

Figure 3. Evaluation of 6OXY-TT in rats.

A) oxycodone-specific serum antibody titers in rats immunized with 6OXY-TT using alum s.c. or Freund’s i.p.; and B) their ability in preventing oxycodone-induced antinociception, shown as % decrease compared to TT control group. Statistical symbols: * p< 0.05 compared to TT control.

Figure 4. Evaluation of GMP grade dKLH in rats.

A) oxycodone-specific serum antibody titers in rats immunized with 6OXY-nKLH or 6OXY-dKLH using alum s.c. or Freund’s i.p.; B) their ability in preventing oxycodone-induced antinociception; C) their effect on oxycodone serum concentration; and D) vaccination effect in preventing brain distribution. Percent (%) decrease compared to nKLH control is shown on top of each group. Statistical symbols: * p< 0.05, **p< 0.01 and *** p< 0.001 compared to KLH control. Brackets to indicate between groups differences.

Figure 5. MPLA decreases immunogenicity of 6OXY-nKLH vaccine.

Oxycodone-specific serum antibody titers in BALB/c mice immunized with alum adjuvant alone, MPLA alone or in combination with alum. Statistical symbols: *** p< 0.001 compared to 6OXY-nKLH in alum s.c. as control.

Figure 6. MPLA decreases immunogenicity of 6OXY-TT vaccine.

A) Oxycodone-specific and TT-specific serum IgG antibody titers in mice vaccinated s.c. with 6OXY-TT in alum or MPLA adjuvants; B) Number of 6OXY-specific antibody secreting B cells (ASC) in mice immunized s.c. with 6OXY-TT using alum or MPLA; and C) Relationship between oxycodone-specific IgG titers and 6OXY-specific antibody secreting B cells including all subjects immunized with 6OXY-TT. Statistical symbols: * p< 0.05 and **p< 0.01 between groups.

Figure 7. Evaluation of 6OXY-nKLH in different mouse strains.

The immunogenicity of 6OXY-nKLH in C57Bl/10ScNJ TRL4^-/- mice was reduced versus C57Bl/10ScSnJ wt controls. A) Oxycodone-specific serum IgG antibody titers in mice vaccinated s.c. with 6OXY-nKLH in alum adjuvant; and B) Number of 6OXY-specific antibody secreting B cells in C57Bl/10ScNJ TRL4^-/- and C57Bl/10ScSnJ wt mice immunized s.c. with 6OXY-nKLH in alum. Statistical symbols: * p< 0.05 and **p< 0.01 compared to C57Bl/10ScSnJ wt control mice.