Bivalent Conjugate Vaccine Induces Dual Immunogenic Response That Attenuates Heroin and Fentanyl Effects in Mice

Abstract

Opioid use disorders and fatal overdose due to consumption of fentanyl-laced heroin remain a major public health menace in the United States. Vaccination may serve as a promising potential remedy to combat accidental overdose and to mitigate the abuse potential of opioids. We previously reported the heroin and fentanyl monovalent vaccines carrying, respectively, a heroin hapten, 6-AmHap, and a fentanyl hapten, para-AmFenHap, conjugated to tetanus toxoid (TT). Herein, we describe the mixing of these antigens to formulate a bivalent vaccine adjuvanted with liposomes containing monophosphoryl lipid A (MPLA) adsorbed on aluminum hydroxide. Immunization of mice with the bivalent vaccine resulted in IgG titers of >10⁵ against both haptens. The polyclonal sera bound heroin, 6-acetylmorphine, morphine, and fentanyl with dissociation constants (K_d) of 0.25 to 0.50 nM. Mice were protected from the anti-nociceptive effects of heroin, fentanyl, and heroin +9% (w/w) fentanyl. No cross-reactivity to methadone and buprenorphine was observed in vivo. Naloxone remained efficacious in immunized mice. These results highlighted the potential of combining TT-6-AmHap and TT-para-AmFenHap to yield an efficacious bivalent vaccine that could ablate heroin and fentanyl effects. This vaccine warrants further testing to establish its potential translatability to humans.

Figure 1

Structure of drugs and conjugates and the research strategy implemented in this study. (a) Chemical structures of heroin, 6-AM, morphine, and fentanyl. (b) Structures of TT-6-AmHap and TT-para-AmFenHap conjugates. The 6-AmHap and para-AmFenHap haptens and the NHS-(PEG)₂-maleimide cross-linker [SM(PEG)₂] linker are depicted in red, blue, and black, respectively. (c) Research strategy: TT-6-AmHap (10 μg) and TT-para-AmFenHap (10 μg) conjugates were mixed with ALF43 and Alhydrogel (ALF43A) adjuvant, and injected i.m. to female Balb/c mice. Adjuvant doses were the same in monovalent and bivalent formulations. The ability of the serum IgG to sequester the opioids in vitro and block their anti-nociceptive effects in vivo was tested.

Figure 2

Immune response to haptens. Mice (n = 10 per group) were immunized at weeks 0, 3, 6, and 14 with the monovalent or bivalent vaccine formulation. Monovalent formulation used 10 μg of antigen. Bivalent vaccine was composed of 10 μg TT-para-AmFenHap and 10 μg TT-6-AmHap. Immunogenicity was evaluated on sera collected on week 16 using ELISA with the indicated coating antigen: (a) animal study timeline; hapten-specific IgG end point titers to (b) BSA-6-AmHap and (c) BSA-para-AmFenHap. Data shown are mean ± SEM. Statistical differences were tested using Mann–Whitney nonparametric t-test (**** p < 0.0001; **, p < 0.005; *, p < 0.05).

Figure 3

Serum sequestration of drugs in vitro. Pooled serum samples from indicated weeks were diluted with a buffer that contained 5 nM of indicated drugs and dialyzed against buffer in an equilibrium dialysis plate. Sodium fluoride (3–4 mg/mL) was added to the buffer to impede heroin degradation. Drug concentrations in the sample and buffer chambers were determined after 24 h, and fraction bound was calculated. Serum binding from monovalent immunization: (a) Fentanyl from TT-para-AmFenHap group and (b) 6-AM from TT-6-AmHap group. Serum binding from bivalent immunization: (c) fentanyl, (d) 6-AM, (e) heroin, and (f) morphine. Data shown are mean ± SEM of triplicate determinations.

Figure 4

Temporal antibody affinity maturation. Pooled serum samples at indicated weeks from mice immunized with monovalent or bivalent vaccines were diluted with a buffer that contained 5 nM of isotopically labeled tracer and dialyzed against buffer in an ED plate. Drug concentrations in the sample and buffer chambers were determined after 24 h, and fraction bound was calculated: (a) TT-6-AmHap monovalent and bivalent vaccine-induced antibodies binding to 6-AM, (b) TT-para-AmFenHap monovalent and bivalent vaccine-induced antibodies binding to fentanyl. The K_d values were calculated as detailed in the Methods section. Data shown are mean ± SEM of triplicate determinations. Statistical significance was determined using the two-tailed, unpaired t test. *, p < 0.05.

Figure 5

Vaccine efficacy against heroin and fentanyl-induced thermal anti-nociception. Mice (n = 10 per group) were challenged s.c. with an increasing dose of either heroin, fentanyl, or 9% (w/w) fentanyl in heroin mix. The anti-nociceptive effects were assessed by hotplate assay 15 min after drug dosing and reported as %MPE: (a) heroin challenge, (b) fentanyl challenge, and (c) heroin + 9% (w/w) fentanyl challenge. Data shown are mean ± SEM. Blue and red arrows depict mice (n = 10) challenged with a single dose of either heroin or fentanyl to measure in vivo cross-reactivity.

Figure 6

Cross-reactivity of bivalent vaccine to therapeutics in vivo. Mice (n = 10 per group) were challenged s.c. with increasing doses of either buprenorphine or methadone. The anti-nociceptive effects were assessed by hotplate assay 15 min after drug dosing and reported as %MPE: (a) buprenorphine, (b) methadone, and (c) experimental flow to assess the cross-reactivity of naloxone in vivo. Mice (n = 10 per group) were challenged s.c. with 0.50 mg/kg heroin with 0.05 mg/kg fentanyl; 15 min afterwards, mice received 0.1 mg/kg naloxone, s.c. The thermal nociception was assessed 5 and 20 min post-naloxone. (d) Anti-nociceptive effects of heroin + 9% (w/w) fentanyl in the presence or absence of naloxone. Data shown are mean ± SEM. Statistical significance (naloxone vs saline group) was determined using a two-tailed, unpaired t-test. ***, p < 0.0005, ****, p < 0.0001. Nlx, naloxone, Sal, saline.