Two doses of Qβ virus like particle vaccines elicit protective antibodies against heroin and fentanyl

Abstract

Opioid overdoses and opioid use disorder (OUD) are major public health concerns. Current treatment approaches for OUD have failed to slow the growth of the opioid crisis. Opioid vaccines have shown pre-clinical success in targeting multiple different opioid drugs. However, the need for many immunizations can limit their clinical implementation. In this study, we investigate the development of novel opioid vaccines by independently targeting fentanyl and the active metabolites of heroin using a bacteriophage virus-like particle (VLP) vaccine platform. We establish the successful conjugation of haptens to bacteriophage Qβ VLPs and demonstrate immunogenicity of Qβ-fentanyl, Qβ-morphine, and Qβ-6-acetylmorphine in animal models after one or two immunizations. We show that these vaccines elicit high-titer, high-avidity, and durable antibody responses. Moreover, we reveal their protective capacities against heroin or fentanyl challenge after two immunizations. Overall, these findings establish Qβ-VLP conjugated vaccines for heroin and fentanyl as promising opioid vaccine candidates.

Fig. 1. Hapten target design and conjugation to Qβ coat protein.

A Hapten targets are chemically conjugated to Qβ VLPs using the bifunctional chemical crosslinker SMPH. Yellow dots indicate surface-exposed lysine residues on Qβ VLPs available for conjugation. Morphine-(Gly)₄ Cys is shown as a representative example; all haptens are conjugated in the same manner. B Hapten design of 6-acetylmorphine (6-AM) and morphine-(Gly)₄ Cys targets. Successful conjugation shown by Coomassie-stained SDS-PAGE. Unconjugated Qβ coat protein has a MW of 14 kDa. The banding pattern of migration indicates 1, 2, or 3 copies of hapten attached per coat protein. C Fentanyl hapten targets using various linkers for attachment. Coomassie-stained SDS-PAGE shows successful conjugation as in (B). PDB ID 7LHD was used as the structure for Qβ VLP in (A) and custom colored for illustration using iCn3D.

Fig. 2. Qβ-morphine and Qβ-6-acetylmorphine vaccine candidates generate high titer, high avidity, durable antibody responses.

Endpoint dilution IgG titers of vaccine-elicited anti-morphine (A) and anti-6-acetylmorphine (B) antibody responses after two immunizations. Responses generated by immunization with Qβ-morphine, Qβ-6-AM, or Qβ-Combo at day 28 (day 7 post-second immunization) are indicated. Data are expressed as geometric mean titer and SD. No significant differences were observed between sexes; Mann-Whitney test. C Antibody avidity to morphine-BSA or 6-AM BSA targets measured by surface plasmon resonance. Serum Avidity is expressed as binding response units/ K_D. D Endpoint dilution IgG titers of vaccine-elicited anti-morphine antibody responses after a single immunization (day 0) with Qβ-morphine and monitored over 170 days; p = 0.0043, Mann-Whitney test.

Fig. 3. Qβ-fentanyl vaccines generate high-titer antibodies.

Anti-fentanyl endpoint dilution IgG titers generated after immunization with FENBB1, FENBB2, or FENBB3 fentanyl vaccine candidates or with the unconjugated Qβ control. Mice (n = 10, Balb/cJ, male and female) were immunized on days 0 and 21.

Fig. 4. Qβ vaccines against heroin metabolites protect against heroin-induced anti-nociception.

A Anti-nociceptive responses measured by tail-flick assay in Balb/cJ mice immunized with Qβ-morphine, Qβ-6AM, Qβ-Combo, Qβ control, or PBS control challenged with heroin (0.5 mg/kg, s.c.) at 3-weeks post-second immunization and tested over a 90 min time course. % Maximum Possible Effect (%MPE = [(drug – basal response)/(20 s – basal response time)] x 100%). B Tail-flick anti-nociceptive responses analyzed at 30 min post-injection with heroin (0.5 mg/kg, s.c.). *p = 0.0364; ***p = 0.0003; vs. PBS control; Kruskal-Wallis test, GraphPad Prism.

Fig. 5. Qβ-fentanyl vaccines protect against anti-nociception and respiratory depression in mice.

A Anti-nociceptive responses assessed by the tail-flick assay in mice vaccinated with Qβ-fentanyl, Qβ control, or PBS and challenged with fentanyl (0.0625 mg/kg, s.c.) at 3-weeks post-second immunization. % Maximum Possible Effect (%MPE = [(drug – basal response)/(20 s – basal response time)] × 100%). P < 0.05 at t = 15, 30, 60, and 90 compared to Qβ control. p < 0.05 at t = 15, 30 and 60 min compared to PBS control; Tukey’s multiple comparison. B Fentanyl-induced decline in respiratory frequency (breaths per minute; BPM) measured by whole-body plethysmography in vaccinated mice (3-weeks post-second immunization). Baseline recordings were taken for 30 min followed by drug challenge with fentanyl (0.25 mg/kg, s.c.) at t = 30 min and recording for an additional 90 min. Data is expressed as normalized values to baseline recordings obtained prior to drug administration for each animal. C Area under the curve (AUC) analysis of the mean frequency data shown in (B). Percent change is indicated between Qβ control and Qβ-Fentanyl vaccinated mice for males and females. ****p < 0.0001; Unpaired t-test; GraphPad Prism.

Fig. 6. Combined fentanyl and heroin vaccine formulation elicits high-titer antibody responses with some cross-reactivity.

Endpoint dilution IgG titers (geometric mean titer) against fentanyl (A), morphine (B), naltrexone (C), methadone (D), and buprenorphine (E). Mice (Balb/cJ, n = 12, male and female) were immunized twice with Qβ-fentanyl, Qβ-morphine + Qβ-6-AM, or Qβ-fentanyl + Qβ-morphine + Qβ-6-AM (20 μg or 10 μg doses). Antibody titers were assessed at day 28 (day 7 post-second immunization).

Fig. 7. Combined fentanyl and heroin vaccine formulation shows protection against fentanyl-induced anti-nociception.

Fentanyl-induced anti-nociception measured by tail-flick assay in vaccinated mice challenged with fentanyl (0.0625 mg/kg, s.c.) at 3 weeks post-second immunization. % Maximum Possible Effect (%MPE = [(drug – basal response)/(20 s – basal response time)] × 100%). Responses at 30 min post-injection are shown. ****p < 0.0001; Dunn’s multiple comparison test; GraphPad Prism.