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. 2024 Jan 25;42(3):471-480.
doi: 10.1016/j.vaccine.2023.12.077. Epub 2023 Dec 29.

A bacteriophage virus-like particle vaccine against oxycodone elicits high-titer and long-lasting antibodies that sequester drug in the blood

Isabella G Romano  1 , Susan B Core  1 , Naomi R Lee  2 , Curtis Mowry  3 , Koen K A Van Rompay  4 , Yumei Huang  5 , Bryce Chackerian  1 , Kathryn M Frietze  6
Affiliations

A bacteriophage virus-like particle vaccine against oxycodone elicits high-titer and long-lasting antibodies that sequester drug in the blood

Isabella G Romano et al. Vaccine. .

Abstract

Opioid use disorder (OUD) and opioid overdoses are public health emergencies. In 2021, 80,000 opioid overdose associated deaths were reported in the United States. Despite the availability of treatment strategies, including medications for opioid use disorder (MOUD) and naloxone, opioid overdoses continue to increase at an alarming rate. Opioid vaccines are a novel approach to combat the growing crisis with several candidates recently entering human clinical trials. In this study, we investigated Qβ bacteriophage virus-like particles (VLPs) as a vaccine platform for immunogenic display of oxycodone. A derivative of oxycodone was conjugated to pre-formed Qβ VLPs using a sulfhydryl-amine reactive heterobifunctional crosslinker with high loading of oxycodone. In mice, intramuscular immunization with Qβ-oxycodone elicited high-titer, high-avidity and long-lasting antibody responses. Qβ-oxycodone was also immunogenic after storage at ambient room temperature for over two weeks, demonstrating that the vaccine is highly thermostable. In mice, immunization with Qβ-oxycodone elicited antibodies that sequester oxycodone in the serum, an important mechanism for preventing the adverse effects of opioid activity. Finally, Qβ-oxycodone is immunogenic in nonhuman primates, eliciting serum oxycodone antibodies after intramuscular immunization of rhesus macaques. These data establish Qβ-oxycodone as a promising opioid vaccine candidate.

Keywords: Antibodies; Opioid; Oxycodone; Vaccine; Virus-like particle.

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Conflict of interest statement

Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Bryce Chackerian reports financial support was provided by National Heart Lung and Blood Institute. Kathryn M. Frietze reports financial support was provided by National Center for Advancing Translational Sciences. Kathryn M. Frietze reports financial support was provided by Office of Research Infrastructure Program, Office of The Director, National Institutes of Health to the California National Primate Research Center. Isabella G. Romano reports financial support was provided by National Institute of Drug Abuse. Bryce Chackerian reports a relationship with Metaphore Biotechnologies, Inc. that includes: equity or stocks. Kathryn M. Frietze has patent Virus-like particle vaccines for opioid drugs pending to None. Bryce Chackerian has patent Virus-like particle vaccines for opioid drugs. pending to None. Susan B. Core has patent Virus-like particle vaccines for opioid drugs pending to None. Naomi R. Lee has patent Virus-like particle vaccines for opioid drugs pending to None. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Figure 1.
Figure 1.. Conjugation of oxycodone-(Gly)4-Cys to Qβ VLPs.
A synthetic derivative of oxycodone was created with a peptide linker composed of four glycine residues and a terminal cysteine residue with c-terminal amidation [(Gly)4-Cys-NH2]; simplified as oxycodone-(Gly)4Cys). (A). Oxycodone-(Gly)4-Cys was then chemically conjugated to the surface amines of Qβ VLPs to generate the Qβ-OXY vaccine via a heterobifunctional crosslinker SMPH (B). Conjugation of Oxycodone-(Gly)4-Cys to Qβ coat protein was confirmed by slower migration of the conjugate (Qβ-OXY) by SDS-PAGE and Coomassie staining compared to unconjugated Qβ coat protein. Average 1.2 oxycodone molecules were loaded onto each unit of Qβ coat protein based on the gel shift assay. Molecular weights: Qβ coat protein (14kDa), oxycodone-(Gly)4-Cys (718Da), addition of linker and oxycodone (984 Da).
Figure 2.
Figure 2.. Qβ-OXY elicits high-titer, high-avidity, long-lasting antibodies.
(A) In a pilot experiment, mice (n=5, Balb/c, Male) were immunized on day 0 with Qβ-OXY (10μg or 20μg), unconjugated Qβ control (20μg), unconjugated KLH control (20μg), KLH-OXY (20ug) or PBS control. Blood was collected at various times post-immunization and assessed for antioxycodone IgG by ELISA, with geometric mean titer reported. ** p<0.005, ****p<0.0001 by Sidak’s multiple comparisons test, compared to Qβ control. (B) Male and female BALB/c mice (n=10) received three immunizations (20μg/50μL, days 0, 21, and 42 indicated by arrows) of QβOXY or unconjugated Qβ control. Blood was collected at various times post-immunization and assessed for anti-oxycodone IgG by ELISA, with geometric mean and geometric SD reported. ****p<0.0001 by Sidak’s multiple comparisons test, compared to appropriate Qβ control (C) Urea-based avidity ELISAs were performed to assess avidity of elicited antibodies after each immunization with Qβ-OXY. Line represents mean avidity index (AI) for each group with error bars indicating SEM. Avidity index (AI) = (A4506M Urea)/(A450H2O) **p<0.01, ****p<0.0001, Brown-Forsythe and Welch’s ANOVA test, GraphPad Prism.
Figure 3.
Figure 3.. Cross-reactivity of Qβ-OXY elicited antibodies with off-target opioids.
Serum from male and female BALB/c mice (n=6) were collected after 1 and 2 immunizations with Qβ-OXY and assessed for binding to buprenorphine and methadone. (A) Oxycodone, buprenorphine, and methadone were used to coat ELISA plates and (B) serum endpoint dilution IgG titers were determined (geometric mean titer and geometric SD are indicated by line and error bars). Dotted line indicates Qβ control serum IgG titer to each target antigen. (C) A urea-based avidity ELISA was used to assess avidity of Qβ-OXY elicited antibodies to each target. Avidity index (AI) is calculated as the ratio of absorbance values for urea treated wells compared to water treated control wells. AI= (A4506M Urea)/(A450H2O). Data points show mean titer with error bars indicating SEM.
Figure 4.
Figure 4.. Qβ-OXY elicited responses sequester oxycodone in the serum upon in vivo drug challenge in mice.
At three-weeks post-second immunization, BALB/c mice (n=12, M/F) were challenged with 2mg/kg subcutaneous oxycodone. 30 min post-injection, truck blood was harvested via decapitation to assess drug serum amount. UPLC-MS based analysis of drug in the serum was conducted for Qβ-OXY and Qβ control immunized animals. Reported values are expressed as the ratio of the peak area of oxycodone compared to the peak area of d6-oxycodone internal standard. ****p<0.0005, Kolmogorov-Smirnov test.
Figure 5.
Figure 5.. Qβ-OXY remains immunogenic after storage at room temperature.
Mice (n=4, BALB/c M/F) received a single intramuscular immunization with Qβ-OXY that had been previously placed through two freeze-thaw cycles and maintained at room temperature for two weeks. Serum was collected at various times post-immunization and anti-oxycodone IgG titers were determined by ELISA. Geometric mean titer with geometric SD are reported.
Figure 6.
Figure 6.. Qβ-oxycodone is immunogenic in nonhuman primates.
Male rhesus macaques (n=3) were immunized with Qβ-OXY at day 0, day 21, and day 273. Serum was collected at various times post immunization and assessed for anti-oxycodone IgG by ELISA. Arrows indicate when each immunization was administered. Data points represent geometric mean titer with geometric SD shown by error bars. Dotted line shows anti-oxycodone IgG end point dilution titer for historical Qβ immunized control rhesus macaque. (B) Avidity of IgG elicited by Qβ-OXY after 1 and 2 immunizations was determined by avidity urea-based avidity ELISA. Avidity index (AI) is the calculated as the ratio of absorbance values in urea treated wells compared to water treated control wells(AI= A4506M Urea)/(A450H2O). *p<0.05 paired t-test, p=0.0349
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