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Published in final edited form as: Bioconjug Chem. 2025 Mar 16;36(4):676–687. doi: 10.1021/acs.bioconjchem.4c00548

Bivalent hapten display strategies for conjugate vaccines targeting opioid mixtures containing fentanyl

Carly Baehr 1,*, Rajwana Jahan 2, Ann Gebo 1, Jennifer Vigliaturo 1, Daihyun Song 1, Md Toufiqur Rahman 2, Davide Tronconi 3,4, Aaron Khaimraj 1, Robert Seaman 3, Courtney Marecki 3, Caroline M Kim 3, Stefano Persano 1, Scott P Runyon 2, Marco Pravetoni 3,5,6
PMCID: PMC12090901  NIHMSID: NIHMS2067238  PMID: 40091228

Abstract

Increasingly, street mixtures of opioids are found to contain combinations of synthetic opioids, such as fentanyl with fentanyl analogs, or counterfeit oxycodone containing fentanyl. While anti-opioid immunotherapy has been investigated as a possible approach to address the opioid epidemic, effectiveness of vaccines and antibodies is limited to specific opioids, based on the chemical structure of the haptens used in vaccines. Hence, there is a need for rational design of anti-opioid conjugate vaccines that simultaneously target multiple opioids. Here, four novel haptens were synthesized, designed to elicit antibodies capable of binding to fentanyl and another opioid, including carfentanil, alfentanil, or oxycodone. Haptens were conjugated to CRM carrier protein and formulated with alum adjuvant, and vaccines containing bivalent haptens were compared to admixture vaccines consisting of conjugates targeting the two opioids separately. Rats were immunized with monovalent, admixture, or novel bivalent vaccines, and blockade of opioid effects was assessed against the individual drugs and their mixtures. Opioid-specific antibody titer was measured, and in vivo effects of vaccines were assessed in terms of preventing opioid-induced antinociception and respiratory depression, and opioid distribution to brain. While the bivalent vaccines reduced the effects of some target opioids, the admixture vaccines were more effective against fentanyl/carfentanil and fentanyl/alfentanil mixtures. The bivalent fentanyl/oxycodone vaccine was as effective as the monovalent vaccines against single drug challenge. These results inform design of future vaccines against opioids and other drugs, particularly in the context of vaccines against polysubstance use that require optimization of response against multiple drugs of interest.

Keywords: Conjugate vaccine, multivalent vaccine, immunotherapy, opioid, fentanyl, substance use disorder

Graphical Abstract:

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INTRODUCTION

An estimated 9 million individuals in the US are in need of treatment for opioid use disorder (OUD) annually; however, of these only ~50% receive treatment, and ~25% receive treatment that includes medication for OUD (MOUD).1 Further, individuals with OUD and other substance use disorders (SUD) are at increased risk for opioid-related overdose, and the prevalence of fentanyl and other potent opioids contributes to high rates of drug overdose fatalities, which exceeded 100,000 in the US in 2022.2 Many such overdoses involve combinations of two or more drugs, or drugs contaminated with fentanyl or other adulterants.3 Fentanyl analogs, including those with higher potency such as carfentanil, are increasingly detected in the illicit drug supply.4,5 While many users report intentional co-use of, for example, fentanyl and heroin,6 some reports indicate as many as one-third of individuals with OUD testing positive for fentanyl may have been exposed unintentionally.7 Illicit oxycodone tablets containing non-pharmaceutical fentanyl have been implicated as a major contributor to overdose deaths, especially when the user may be unaware of the presence of fentanyl.8 Similarly, use of stimulants, such as methamphetamine and cocaine, containing fentanyl contributes to the incidence of overdose among opioid-naïve individuals.9

Current MOUD and overdose reversal agents target μ opioid receptors (MOR). Though these treatments are effective, their use is stigmatized among patients and health care providers.10,11 Patients report that strict requirements imposed by treatment programs may make retention in treatment more difficult.12 Logistical barriers, such as insurance status, long travel distance to treatment centers, and limited availability of MOUD at some pharmacies, also contribute to low treatment retention,13,14 and inequality of access to treatment programs across demographic and economic lines has resulted in the burden of the OUD epidemic falling disproportionately on marginalized communities.15 The recent removal of a special certification required since 2000 for prescribing buprenorphine (the X-Waiver) is expected to increase future access to this MOUD, though state-level regulations and other barriers to treatment still exist.16,17

Treatments for OUD that do not require frequent pharmacy visits, and that lack regulatory hurdles due to diversion liability, warrant investigation to ease the burden of OUD management on patients and providers. Immunotherapy using vaccines elicits drug-binding antibodies, which can neutralize target opioids in blood, and prevent distribution to the brain and subsequent interaction with MOR. Such vaccines consist of haptens mimicking the structure of the target drug conjugated to an immunogenic carrier protein. Immunotherapy carries a low perceived risk to patients, has long-lasting effects, and could be combined with MOUD.18,19 Broadly, drug-targeting vaccines have substantial evidence of safety and effectiveness in preclinical models. However, vaccines carry the drawback that elicited antibodies are selective for the structure of the targeted drug, and typically have low cross-reactivity for structurally dissimilar compounds. While this specificity prevents off-target binding of antibodies to MOUD or endogenous opioids, it may also reduce the ability of antibodies to recognize fentanyl analogs and other opioids that carry overdose risk. Hence, development of vaccines that simultaneously target the most prevalent drugs involved in overdose is critical for SUD vaccines to be a viable treatment option.

The goal of this work was to compare conjugate vaccines containing novel bivalent hapten designs, which target multiple drugs with connected haptens on the same linker, to corresponding monovalent and admixture vaccines utilizing the individual haptens. Previously, an oxycodone-targeting vaccine (OXY-sKLH) has been extensively characterized in rodent models, showing evidence of effectiveness in reducing effects of oxycodone.20,21 Previous work on vaccines for OUD also identified lead vaccines against fentanyl (F1-CRM and F6-CRM)22 as well as candidate vaccines against fentanyl analogs including alfentanil (F8-CRM)23 and carfentanil (F13-CRM)24. To compare these strategies for targeting multiple opioids, monovalent conjugate vaccines were compared to four novel bivalent haptens that target two opioids simultaneously, including fentanyl in combination with either carfentanil, alfentanil, or oxycodone. Conjugate vaccines and admixture vaccines were compared in terms of vaccine-elicited titers and blockade of opioid-induced effects in rats, including against individual drugs and drug mixtures. While the novel bivalent vaccines were effective in altering distribution of opioids to brain, the admixture vaccines were more effective and were able to reduce the pharmacological effects of opioid mixtures in rats. These studies extend previous work examining monovalent opioid-targeting vaccines and inform future development of multidrug-targeting vaccines for treatment of OUD involving multiple opioids, such as drug mixtures contaminated with fentanyl.

RESULTS

Synthesis of bivalent haptens and conjugate vaccines targeting multiple opioids.

Novel bivalent haptens F15, F16, F6/8, and F17 were synthesized, and conjugated to CRM carrier protein. The F15 hapten was based on the fentanyl analog 4-phenylfentanyl.25 Whereas carfentanil contains a methoxymethyl ester moiety, 4-phenylfentanyl contains a phenyl substituent, which we hypothesized would allow the binding pockets of elicited antibodies to accommodate a larger functional group at the 4 position and generate cross-reactivity to fentanyl analogs in the carfentanil class. The other novel haptens F16, F6/8, and F17 contained branching linkers that terminated in structures matching their corresponding monovalent haptens. Monovalent CRM conjugates (F1-CRM, F13-CRM, F6-CRM, F8-CRM) showed haptenation ratios (HR) between 12.1 and 19.4 (Supplemental Table S1). The HR of OXY-sKLH could not be determined due to the larger molecular weight of sKLH conjugate. Bivalent conjugates showed HR of 14.5 (F15-CRM), 13.1 (F16-CRM), 14.1 (F6/8-CRM), and 9.2 (F17-CRM).

Effects of novel bivalent vaccines against fentanyl and carfentanil in rats.

Two conjugate vaccines containing novel bivalent haptens (F15, F16) were evaluated in comparison to monovalent haptens. In a first experiment, rats (n=6 per group) were immunized with CRM control, F1-CRM or F13-CRM alone, an admixture of F1-CRM and F13-CRM containing a ½ dose of each monovalent conjugate, or the novel F15-CRM (Figure 1A). F1-CRM and the admixture generated the highest fentanyl-specific antibody titer (Figure 1B), and F13-CRM and the admixture generated the highest carfentanil-specific titer (Figure 1E). Rats immunized with F15-CRM produced low fentanyl- and carfentanil-specific titer when measured by ELISA using the F1 and F13 haptens, but antibodies were detected using F15 as the coating antigen for ELISA (Supplemental Figure S1A). Consistent with the previously reported F1-CRM and F13-CRM vaccines alone or in combination,24 F1-CRM alone and the F1-CRM+F13-CRM admixture reduced the effects of fentanyl (Figure 1C–D). F13-CRM reduced the effects of carfentanil on oxygen saturation, but there was no statistically significant effect on carfentanil-induced antinociception (Figure 1F–G). The bivalent vaccine F15-CRM did not significantly reduce the antinociceptive or cardiopulmonary effects of either drug challenge compared to control.

Figure 1. Fentanyl and carfentanil vaccine F15-CRM against individual drugs in rats.

Figure 1.

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A, Structure of F15 hapten, and of F1 and F13 haptens. B, F1 (fentanyl-specific) titer after two (2x, day 21) or four immunizations (4x, day 49). Rats were challenged with 0.1 mg/kg fentanyl s.c. and monitored at 15-minute intervals. C, Fentanyl-induced antinociception measured as latency to respond on a hot plate; D, fentanyl-induced respiratory depression measured as % oxygen saturation by pulse oximetry. E, F13 (carfentanil-specific) titer after two (2x, day 21) or four immunizations (4x, day 49). Rats were challenged with 0.02 mg/kg carfentanil s.c. and monitored at 15-minute intervals. F, Carfentanil-induced antinociception measured as latency to respond on a hot plate; G, carfentanil-induced respiratory depression measured as % oxygen saturation by pulse oximetry. Data are mean +/− SEM; *p<0.05, **p<0.01 compared to CRM control.

In a second experiment, rats were immunized with CRM control, an admixture of F1-CRM and F13-CRM, or the novel F16-CRM (Figure 2A). Rats immunized with the admixture vaccine generated high fentanyl-specific and carfentanil-specific titers (Figure 2B, 2E), and rats immunized with F16-CRM generated lower levels of antibodies measured against F1 and F13, but as with F15-CRM, antibodies were detected using F16 as the coating antigen (Supplemental Figure S1B). As in the first experiment, the F1-CRM + F13-CRM admixture blocked the effects of fentanyl (Figure 2C–D). Neither F16-CRM nor the admixture blocked the effects of carfentanil at a dose of 0.02 mg/kg (Supplemental Figure S2). Because rats immunized with the admixture vaccine showed oxygen saturation values of <80% in this challenge, the dose of carfentanil was reduced to 0.01 mg/kg for a subsequent carfentanil challenge. At this dose, the F1-CRM + F13-CRM admixture vaccine reduced the effects of carfentanil on oxygen saturation (Figure 2F–G). The effect of the bivalent vaccine F16-CRM was not statistically significant in comparison to control for either individual drug challenge.

Figure 2. Fentanyl and carfentanil vaccine F16-CRM against individual drugs in rats.

Figure 2.

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A, Structure of F16 hapten, with structure of F1 shown in red and F13 shown in blue. B, F1 (fentanyl-specific) titer after two (2x, day 21) or four immunizations (4x, day 49). Rats were challenged with 0.1 mg/kg fentanyl s.c. and monitored at 15-minute intervals. C, Fentanyl-induced antinociception measured as latency to respond on a hot plate; D, fentanyl-induced respiratory depression measured as % oxygen saturation by pulse oximetry. E, F13 (carfentanil-specific) titer after two (2x, day 21) or four immunizations (4x, day 49). Rats were challenged with 0.01 mg/kg carfentanil s.c. and monitored at 15-minute intervals. F, Carfentanil-induced antinociception measured as latency to respond on a hot plate; G, carfentanil-induced respiratory depression measured as % oxygen saturation by pulse oximetry. Data are mean +/− SEM; *p<0.05, **p<0.01 compared to CRM control.

After the individual drug challenges were completed, rats were challenged with a mixture of fentanyl and carfentanil to test efficacy against drug mixture and to evaluate the effect of vaccines on drug distribution. The F13-CRM vaccine alone and the F1-CRM + F13-CRM admixture significantly reduced the effects of the drug mixture (Figure 3A–B, E–F), and all vaccines significantly reduced the brain:serum distribution ratio of both drugs (Figure 3C–D, G–H). Interestingly, the bivalent F15-CRM did not produce a statistically significant difference from control in the antinociception and oxygen saturation tests, but reduced fentanyl and carfentanil distribution to brain similar to F13-CRM. Similarly, the bivalent F16-CRM reduced fentanyl and carfentanil distribution to brain, but was not as effective as the admixture vaccine.

Figure 3: Fentanyl and carfentanil vaccines F15-CRM and F16-CRM against fentanyl/carfentanil mixture.

Figure 3:

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For testing F15-CRM vaccine, rats were challenged with a mixture of 0.05 mg/kg fentanyl and 0.01 mg/kg carfentanil. A, Fentanyl/carfentanil mixture-induced antinociception measured as latency to respond on a hot plate. B, Fentanyl/carfentanil mixture-induced respiratory depression measured as % oxygen saturation by pulse oximetry. Data are mean +/− SEM. After the last measurement, distribution of each drug to brain compared to serum was measured. C, Ratio of fentanyl in brain vs serum; D, ratio of carfentanil in brain vs serum. Data are mean +/− SD. For testing F16-CRM vaccine, rats were challenged with a mixture of 0.05 mg/kg fentanyl and 0.005 mg/kg carfentanil. E, Fentanyl/carfentanil mixture-induced antinociception measured as latency to respond on a hot plate. F, Fentanyl/carfentanil mixture-induced respiratory depression measured as % oxygen saturation by pulse oximetry. Data are mean +/− SEM. After the last measurement, distribution of each drug to brain compared to serum was measured. G, Ratio of fentanyl in brain vs serum; H, ratio of carfentanil in brain vs serum. Data are mean +/− SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to CRM control.

Effects of a bivalent vaccine against fentanyl and alfentanil in rats.

Conjugate vaccines were tested against the combination of fentanyl and alfentanil. F6-CRM was chosen as the fentanyl-targeting vaccine for this experiment because it had a similar linker position to the alfentanil-targeting vaccine, F8-CRM (Figure 4A).22,23 For evaluation of vaccines against fentanyl and alfentanil, rats (n=6 per group) were immunized with CRM control, F6-CRM or F8-CRM alone, an admixture of F6-CRM and F8-CRM, or the novel bivalent F6/8-CRM. F6-CRM and the admixture generated high fentanyl-specific antibody titer, while F8-CRM and the admixture generated high alfentanil-specific titer (Figure 4B, 4E). The bivalent conjugate vaccine generated detectable antibody titers against both drugs. The F6-CRM vaccine and the F6-CRM + F8-CRM admixture vaccine reduced fentanyl-induced antinociception in rats (Figure 4C), while the F8-CRM and the F6-CRM + F8-CRM admixture vaccine reduced alfentanil-induced antinociception (Figure 4F); effects on oxygen saturation were not significant (Figure 4D, 4G). The novel bivalent vaccine F6/8-CRM reduced the effects of fentanyl, but was not significantly different from CRM control in the alfentanil challenge.

Figure 4: Fentanyl and alfentanil vaccines against individual drugs in rats.

Figure 4:

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A, Structure of F6/8 hapten, with structure of F6 shown in red and F8 shown in blue. B, F6 (fentanyl-specific) titer after two (2x, day 21) or four immunizations (4x, day 49). Rats were challenged with 0.1 mg/kg fentanyl s.c. and monitored at 15-minute intervals. C, Fentanyl-induced antinociception measured as latency to respond on a hot plate; D, fentanyl-induced respiratory depression measured as % oxygen saturation by pulse oximetry. E, F8 (alfentanil-specific) titer after two (2x, day 21) or four immunizations (4x, day 49). Rats were challenged with 0.25 mg/kg alfentanil s.c. and monitored at 15-minute intervals. F, Alfentanil-induced antinociception measured as latency to respond on a hot plate; G, alfentanil-induced respiratory depression measured as % oxygen saturation by pulse oximetry. Data are mean +/− SEM; *p<0.05, **p<0.01, ****p<0.0001 compared to CRM control.

Rats were then challenged with a mixture of fentanyl and alfentanil. In this experiment, F6-CRM, F8-CRM, and the admixture vaccine reduced the effects of the drug mixture, but the F6-CRM + F8-CRM admixture vaccine was the most effective (Figure 5A–B). The distribution of fentanyl and alfentanil to brain vs serum was measured after the challenge. All vaccines significantly reduced fentanyl distribution to brain, and F6-CRM and the admixture vaccine produced the largest reduction in fentanyl distribution compared to F8-CRM and the F6/8-CRM bivalent vaccine (Figure 5C). Only F8-CRM and the F6-CRM + F8-CRM admixture vaccine reduced alfentanil distribution to brain vs serum (Figure 5D).

Figure 5: Fentanyl and alfentanil vaccines against fentanyl/alfentanil mixture.

Figure 5:

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Rats were challenged with a mixture of 0.05 mg/kg fentanyl and 0.125 mg/kg alfentanil. A, Fentanyl/alfentanil mixture-induced antinociception measured as latency to respond on a hot plate. B, Fentanyl/alfentanil mixture-induced respiratory depression measured as % oxygen saturation by pulse oximetry. Data are mean +/− SEM. After the last measurement, distribution of each drug to brain compared to serum was measured. C, Ratio of fentanyl in brain vs serum; D, ratio of alfentanil in brain vs serum. Data are mean +/− SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to CRM control.

Effects of a bivalent vaccine against fentanyl and oxycodone in rats.

The combination of F1-CRM and OXY-sKLH vaccines was previously found to be as effective as either individual vaccine when co-administered.26 For evaluation of vaccines against fentanyl and oxycodone, rats (n=6 per group) were immunized with CRM control, F1-CRM or OXY-sKLH alone, or the novel F17-CRM bivalent vaccine (Figure 6A). Fentanyl-specific titer was highest in rats immunized with F1-CRM and F17-CRM (Figure 6B), and oxycodone-specific titer was highest in rats immunized with OXY-sKLH and F17-CRM, while no anti-oxycodone response was detected in F1-CRM immunized rats (Figure 6E). In initial individual drug challenges, a dose of 2.25 mg/kg oxycodone did not produce significant antinociception or respiratory depression in all vaccine groups (Supplemental Figure S3), whereas F1-CRM and the F17-CRM bivalent vaccine both reduced the effects of fentanyl (Figure 6C–D). A subsequent oxycodone challenge was performed using 4.5 mg/kg oxycodone; in this experiment, the F17-CRM bivalent vaccine blocked the effects of oxycodone, while the OXY-sKLH and F1-CRM showed no difference compared to control (Figure 6F–G).

Figure 6: Fentanyl and oxycodone vaccines against individual drugs in rats.

Figure 6:

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A, Structure of F17 hapten, with structure of F1 shown in red and OXY shown in blue. B, F1 (fentanyl-specific) titer after two (2x, day 21) or four immunizations (4x, day 49). Rats were challenged with 0.1 mg/kg fentanyl s.c. and monitored at 15-minute intervals. C, Fentanyl-induced antinociception measured as latency to respond on a hot plate; D, fentanyl-induced respiratory depression measured as % oxygen saturation by pulse oximetry. E, OXY (oxycodone-specific) titer after two (2x, day 21) or four immunizations (4x, day 49). Rats were challenged with 4.5 mg/kg oxycodone s.c. and monitored at 15-minute intervals. F, Oxycodone-induced antinociception measured as latency to respond on a hot plate; G, oxycodone-induced respiratory depression measured as % oxygen saturation by pulse oximetry. Data are mean +/− SEM; *p<0.05, **p<0.01, ****p<0.0001 compared to CRM control.

Rats were then challenged with a mixture of fentanyl and oxycodone. OXY-sKLH, F1-CRM, and the F17-CRM bivalent vaccine all reduced antinociception induced by the drug mixture, though only F1-CRM blocked the effects of the drug mixture on oxygen saturation (Figure 7A–B). Both F1-CRM and F17-CRM reduced fentanyl distribution to brain vs serum, and both OXY-sKLH and F17-CRM reduced oxycodone distribution (Figure 7C–D).

Figure 7: Fentanyl and oxycodone vaccines against fentanyl/oxycodone mixture.

Figure 7:

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Rats were challenged with a mixture of 0.05 mg/kg fentanyl and 2.25 mg/kg oxycodone. A, Fentanyl/oxycodone mixture-induced antinociception measured as latency to respond on a hot plate. B, Fentanyl/oxycodone mixture-induced respiratory depression measured as % oxygen saturation by pulse oximetry. Data are mean +/− SEM. After the last measurement, distribution of each drug to brain compared to serum was measured. C, Ratio of fentanyl in brain vs serum; D, ratio of oxycodone in brain vs serum. Data are mean +/− SD. ***p<0.001, ****p<0.0001 compared to CRM control.

DISCUSSION

Effective OUD treatment often requires a combination of strategies, which may include behavioral therapy and MOUD. Longer treatment retention is associated with better outcomes, whereas discontinuation of MOUD may lead to withdrawal and increased risk of overdose.27 Adding vaccines that elicit production of long-lasting antibodies to the arsenal of available OUD therapies could provide an effective tool to mitigate the risk of overdose, especially with the increasing presence of fentanyl and other highly potent opioids in the drug supply. Because treatment of overdose is complicated by the presence of multiple substances, exploring bivalent vaccines as a strategy to broaden the range of targeted opioids could increase the utility of immunotherapeutics currently under investigation.

The present study sought to compare bivalent strategies to elicit protective antibody titers against drug mixtures containing fentanyl. The monovalent fentanyl,22 carfentanil,24 and oxycodone20,21,28 vaccines used here were previously shown to be effective in rats; the alfentanil-targeting F8-CRM showed limited effectiveness,23 though the blood/brain distribution of alfentanil was not examined in that study as it was here. Co-administration of fentanyl-carfentanil vaccines,24 fentanyl-oxycodone vaccines,26 and pentavalent vaccines (targeting fentanyl, carfentanil, oxycodone, heroin and methamphetamine)26 maintained similar effectiveness to the monovalent vaccines, supporting investigation of multivalent immunization strategies for SUD.

Here, the admixture vaccines were consistently more effective than vaccines containing bivalent haptens. HR was similar among conjugates, though F16, F6/8, and F17 combine two molecules per hapten, so the effective HR is double the listed value in Supplemental Table S1. F17-CRM yielded the lowest HR, 9.2 (Supplemental Table S1). Despite this, in the individual drug challenges, of the tested bivalent vaccines only F17-CRM targeting fentanyl and oxycodone was able to significantly reduce the effects of the individual opioids in vivo. The oxycodone vaccine utilized sKLH carrier as opposed to CRM carrier, so direct comparison with F17-CRM may not be possible; however, previous studies suggest that OXY-sKLH and OXY-CRM produce similar results.29 Surprisingly, the F17-CRM vaccine was more effective than OXY-sKLH alone in preventing the effects of the oxycodone challenge, though F17-CRM elicited oxycodone titers were lower than OXY-sKLH elicited titers. This may be due to polyclonal antibodies against the bivalent hapten not recognizing the immobilized monovalent oxycodone hapten in the context of the ELISA assay, thus reducing the apparent titer.

All monovalent, admixture, and bivalent vaccines tested were able to elicit antibodies that altered the distribution of at least one target opioid to brain. Interestingly, the fentanyl brain:serum ratios in the control groups differed slightly across experiments with different drug combinations: ~1.5 for fentanyl/carfentanil, 0.8 for fentanyl/alfentanil, and 6.0 for fentanyl/oxycodone. This may be due to drug-drug interactions with fentanyl, as the doses of alfentanil and oxycodone were higher than the fentanyl dose, whereas carfentanil was administered at a lower dose than fentanyl. Notably, the monovalent vaccines that target opioids with some similarity in structure, i.e. fentanyl/carfentanil and fentanyl/alfentanil, affected distribution of the non-targeted opioid. That is, F1-CRM alone reduced carfentanil distribution, F13 alone reduced fentanyl distribution, and F8-CRM reduced fentanyl distribution. On the other hand, F6-CRM and F6/8-CRM had no significant impact on alfentanil distribution, although F8-CRM alone or admixed with F6-CRM showed only modest effect. F1-CRM did not affect oxycodone distribution, and OXY-sKLH did not affect fentanyl distribution, which is expected based on the divergent structures of fentanyl and oxycodone.

The bivalent haptens examined here varied in several critical respects: whether the targeted drugs have similar (fentanyl vs carfentanil) vs divergent (fentanyl vs oxycodone) structures, and the length of the inter-hapten spacer. Whereas F15 contained a single moiety intended to simultaneously mimic fentanyl and carfentanil, with no spacer, F16 contained a spacer 6 atoms in length; F6/8, 9 atoms; and F17, 14 atoms. It is possible that this inter-hapten spacer length is a major factor in determining vaccines’ ability to engage B cells and initiate adaptive immune response.

In the context of SUD vaccines, strategies for obtaining robust immune response against multiple drug targets have included: masking of structural differences between drugs (e.g. heroin vs morphine) through strategic linker placement;30 multivalent (co-administered or admixture) vaccines combining two or more conjugates with different haptens;31–34 and vaccines with bivalent (or chemically contiguous) haptens containing two or more drug moieties per linker.35,36 While such strategies have broadly been shown to be effective in terms of generating detectable anti-drug titers, direct comparison of these strategies can be difficult due to studies using different haptens, linker chemistry, carriers, and adjuvants. Similarly, bivalent strategies have been explored as a strategy to improve immune response to vaccines against a single drug target; studies of anti-nicotine vaccines found that combining vaccines utilizing haptens with different linker positions may improve overall nicotine-specific titer.37,38 On the other hand, hapten clustering strategies have yielded mixed results. Clustering of identical nicotine haptens on the same linker generated vaccines with similar effectiveness to monovalent vaccines, though the overall hapten density was lower;39 and a peptide vaccine containing one methamphetamine hapten per peptide out-performed the bivalent peptide.40 Overall, these studies may imply that clustering of identical haptens may act similarly to increasing the net hapten “dose”, whereas pairing non-identical haptens may increase the maximum achievable response by engaging broader B cell populations.41 Our observation that F17 was the most effective bivalent design is broadly consistent with this conclusion, though further study would be required to determine to what extent F1/F13 and F6/F8 combinations engage overlapping B cell populations.

Overall, the results of this study indicate several factors that may inform design of multivalent vaccines for SUD in the context of polydrug use. Advancement of vaccines for SUD toward clinical development will further require careful optimization of hapten chemistry and vaccine formulation. A limitation of the present work is that all vaccine formulations used the relatively weak alum adjuvant; future studies of bivalent vaccines could benefit from inclusion of more potent adjuvants in experimental vaccines. Generally, vaccines for SUD have performed well in preclinical models, but yielded less promising results in clinical trials, usually due to low antibody response.18,42 Because effectiveness of SUD vaccines requires high concentration and affinity of circulating antibodies, current work in the field has focused on selection of adjuvants to enhance antibody response and on identification of biomarkers to select patient populations most likely to benefit from immunotherapy approach.43–45 Such strategies can be combined with these bivalent vaccine approaches to aid translation of SUD vaccines to protect from drug mixtures containing fentanyl.

MATERIALS AND METHODS

Conjugation to carrier proteins.

The series of F/FA-targeting haptens (F1, F13, F6, F8, F15, F16, F17, and F6/8) were synthesized (see Supporting Information) and conjugated to diphtheria toxoid cross-reactive material (CRM) as previously described to generate F1-CRM, F13-CRM, F6-CRM, F8-CRM, F15-CRM, F16-CRM, F17-CRM, and F6/8-CRM.22–24 The oxycodone-targeting hapten (OXY) was conjugated to sKLH as described.46 Briefly, the haptens were dissolved in a 0.1 M 2-(N-morpholino) ethanesulfonic acid (MES) buffer with 250 mM sucrose as a stabilizing agent at a pH of 5.0. For F16, F6, F8, and F6/8, 10% DMSO was added to a concentration of 5.2 mM. Carbodiimide cross-linking chemistry was achieved by activating the haptens with 208 mM N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC). After 15 minutes of stirring at room temperature, CRM was added to a final concentration of 2.8 mg/mL and stirred for 3 hours at room temperature. To terminate the reaction, the conjugate mixture was washed with PBS buffer at pH 7.2 using an Amicon filter (MerckMillipore, Burlington, MA) with a 30 kDa molecular weight cutoff, then reconstituted with 250 mM sucrose in PBS buffer at pH 7.2 to a final concentration of 2.5 mg/mL, and stored at 4°C.

Drugs.

Pharmaceutical grade fentanyl citrate and alfentanil HCl were obtained from Boynton Pharmacy. Oxycodone HCl was obtained from the University of Washington Pharmacy. Carfentanil HCl was obtained from the NIDA Drug Supply Program. Drug doses and concentrations are expressed as weight of the free base.

Safety.

Researchers adhered to all safety protocols recommended by respective regulatory authorities. Extreme caution was followed while handling and manipulating the materials including opioids and opioid-based haptens to avoid exposure and inhalation. Opioid antagonist was stored in an easily accessible location and all advance-stage materials were stored in a secured vault. No unintended exposure or unexpected hazard were encountered during these processes, and thus no unusual exposure risk and hazards have been identified as a result of this work.

Animals.

All animal studies were approved by the Institutional Animal Care and Use Committees of the University of Minnesota and University of Washington. Male Sprague Dawley rats (n=6/group), 250 g on arrival, were housed in pairs under standard conditions with ad libitum access to food and water, on a 14/10 light/dark cycle. Experiments occurred during the light cycle, and animals were habituated to the experimentation room for 1 hour prior to drug challenges.

Immunization.

For vaccine formulation, conjugates were adsorbed on alum adjuvant (Alhydrogel). Each vaccine dose contained 60 μg conjugate and 90 μg alum, diluted to 150 μL total volume with sterile PBS buffer. For admixture vaccines, individual conjugates were adsorbed separately on alum and then mixed in equal amounts. Negative control vaccines consisted of 60 μg unconjugated CRM adsorbed on 90 μg alum for the experiments with F15, F16, and F6/8, and consisted of 90 μg alum alone for the experiment with F17. Rats were immunized four times i.m. with 150 μL of vaccine on days 0, 14, 28, and 42. Serum was collected one week after the 2nd and 4th vaccinations, on days 21 and 49. For experiments with F16-CRM and F17-CRM, rats were boosted an additional time after initial single-drug challenges, on day 81 and on day 65, respectively.

Determination of anti-opioid titer.

High-binding polystyrene microtiter plates were coated overnight with haptens conjugated to BSA, 0.05 μg/mL in carbonate buffer, pH 8.5. Serum samples were applied to coated plates and serially diluted, starting at 1:200 initial dilution, and incubated for 2 hours. Then, plates were washed, and incubated overnight with goat anti-rat secondary antibody, HRP conjugate. HRP activity was quantitated with OPD substrate, and titer was calculated as EC50 of dilution value.

Drug challenges.

Starting 1 week after the 4th vaccination, rats were challenged with fentanyl or other opioid (carfentanil, alfentanil, or oxycodone), alternating the order of challenges such that 3 rats per group received fentanyl first or other opioid first. Drugs were prepared in sterile PBS and delivered subcutaneously with 1 mL/kg injection volume. Drug doses were fentanyl 0.1 mg/kg, carfentanil 0.02 mg/kg, alfentanil 0.25 mg/kg, and oxycodone 2.5 mg/kg as indicated in the figure legends. Rats were monitored for up to 60 minutes for drug-induced antinociception by latency to respond on a hot plate (Columbus Instruments), and for respiratory depression by pulse oximetry (Starr Life Sciences). For the experiment with F16-CRM, the carfentanil challenge was repeated with 0.01 mg/kg carfentanil after the 5th vaccination. For the experiment with F17-CRM, the oxycodone challenge was repeated with 4.5 mg/kg oxycodone after the 5th vaccination. After a one-week washout period following the last challenge, rats received a mixture of opioids with each drug at half the dose given in individual challenges, and were monitored for 30 minutes. Then, rats were euthanized, and serum and brain were collected for analysis of drug concentration.

Determination of opioid concentration in tissue.

Analysis of fentanyl, carfentanil, alfentanil, or oxycodone concentration in brain and serum by liquid chromatography-mass spectrometry (LC-MS) was performed essentially as described.22,24 For sample extraction, 100 μL of sample was used for extraction with the addition of 10μL of internal standard solution (100ng/mL). Standards were prepared with 10μL of stock calibrator solution in 90μL of fetal bovine serum. Cold acetonitrile (300 μL) was added to precipitate protein and samples were centrifuged at 8,600 x g for 10 min. Supernatant was transferred to a 96 square deep-well plate, evaporated to 100μL with a MiniVap sample concentrator (Porvair Sciences, Wrexham, UK) and then diluted with 300μL of 2% phosphoric acid. Extraction was performed using a Bond Elut PCX 96 round, 1mL, 30mg plate (Agilent, Santa Clara, CA) pre-washed with 500μL methanol followed by 500μL. The wells were washed with 300μL of 2% formic acid followed by 300μL of 50% methanol:50% acetonitrile. The extraction plate was dried on a positive pressure manifold and samples eluted into a new deep well round bottom collection plate with three washes of 125μL of 5% ammonium hydroxide in 50% methanol:50% acetonitrile. Eluate was evaporated until almost dry on the sample concentrator and reconstituted in 100μL mobile phase A (water, 5mM ammonium formate, 0.01% formic acid).

LCMS/MS conditions.

Samples (2 μL) were injected onto a reversed phase Poroshell SB-C18 (2.1 mm × 50 mm i.d., 2.7 μm) column at 55°C. The LCMS/MS system consisted of an Agilent G6470A triple quadrupole with an Infinity II 1290 G7116B Multicolumn Thermostat, G7120A High Speed Quad Pumps, G7267B multisampler. The samples were kept at 4°C during injection. Gradient elution was performed with a mixture of mobile phase A and methanol, 0.01% formic acid (mobile phase B) as follows: 0–0.5 min 5% mobile phase B, 0.5–3.0 min 15 → 50% mobile phase B, 3.0–4.0 min 50 → 95% mobile phase B, 4.0–6.0 95% mobile phase B. The flow rate was kept at a constant 0.40 mL/min and the total run time was 6 min. The auto-sampler needle was multi-washed with 0.01% formic acid in water, 50% acetonitrile:50% methanol, then 70% isopropanol following each sample injection. Electrospray ionization was achieved by Agilent Jet Stream high sensitivity ion source in the positive ion mode. Instrument settings were: gas temperature 325°C, gas flow 9 L/min, nebulizer pressure 40 psi, sheath gas temperature 380°C, sheath gas flow 10 L/min, capillary 2,500 V, and nozzle 0V. Data acquisition and peak integration were interfaced to a computer workstation using Mass Hunter software (Tokyo, Japan). LCMS/MS in the SIM mode was used to identify the appropriate ions to monitor: fentanyl primary 337.2→188.1, secondary 337.2→105.1, fentanyl D5 342.3→105.1, alfentanil 323.2→105.1, alfentanil 323.2→188.1.

Statistical analysis.

All statistical analyses were conducted with Prism v 10 (GraphPad). For antinociception and oxygen saturation, differences over time between groups were analyzed by 2-way ANOVA or mixed model with Dunnett’s multiple comparisons test. Blood/brain distribution ratios were analyzed by one-way ANOVA with Tukey’s multiple comparisons test.

Supplementary Material

bivalent hapten display supplement

Acknowledgements:

This work was supported by NIH under grant UG3 DA048386 (Pravetoni).

Abbreviations:

BSA

Bovine serum albumin

CRM

cross-reactive molecule carrier protein

i.m.

intramuscular

MOR

mu opioid receptor

MOUD

medication for opioid use disorder

OUD

opioid use disorder

OXY

oxycodone hapten

s.c.

subcutaneous

sKLH

subunit keyhole limpet hemocyanin carrier protein

SUD

substance use disorder

Footnotes

Conflict of interest statement: Structures of the novel haptens utilized in this study are the subject of provisional patent disclosures. Pravetoni is the founder of CounterX Therapeutics, Inc. Other authors declare no competing financial interest.

Supporting Information: Additional experimental details with reaction schemes for hapten synthesis; table of conjugate properties; F15- and F16-specific titer data; and carfentanil and oxycodone dose-finding studies (.docx).

Data availability statement:

All source data for the current study are available from the corresponding author on reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

bivalent hapten display supplement
NIHMS2067238-supplement-bivalent_hapten_display_supplement.docx (606KB, docx)

Data Availability Statement

All source data for the current study are available from the corresponding author on reasonable request.

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