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Cited by 15Monoclonal and Polyclonal Antibodies ResearchImmunotherapy and Immune ResponsesOpioid Use Disorder Treatment

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Self-Adjuvanting TLR7/8 Agonist and Fentanyl Hapten Co-Conjugate Achieves Enhanced Protection against Fentanyl Challenge

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Powers, Noah, Massena, Casey, Crouse, Bethany, Smith, Mira, Hicks, Linda, Evans, Jay T., Miller, Shannon, Pravetoni, Marco, Burkhart, David·2023·Bioconjugate Chemistry
DOI 10.1021/acs.bioconjchem.3c00347PMC PMC10587865
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Abstract

High Resolution Image Download MS PowerPoint Slide Currently approved pharmacotherapies for opioid use disorders (OUDs) and overdose reversal agents are insufficient to slow the spread of OUDs due to the proliferation of fentanyl. This is evident in the 31% rise in drug overdose deaths from 2019 to 2022, with rates increasing from 21.6 to 28.3 overdoses per 100,000 deaths. Vaccines are a potential alternative or adjunct therapy for the treatment of several substance use disorders (nicotine, cocaine) but have shown limited clinical success due to suboptimal antibody titers. In this study, we demonstrate that coconjugation of a Toll-like receptor 7/8 (TLR7/8) agonist (UM-3006) alongside a fentanyl-based hapten (F 1 ) on the surface of the carrier protein cross-reactive material 197 (CRM) significantly increased generation of high-affinity fentanyl-specific antibodies. This demonstrated enhanced protection against fentanyl challenges relative to an unconjugated (admix) adjuvant control in mice. Inclusion of aluminum hydroxide (alum) adjuvant further increased titers and enhanced protection, as determined by analysis of fentanyl concentration in serum and brain tissue. Collectively, our findings present a promising approach to enhance the efficacy of antiopioid vaccines, underscoring the need for extensive exploration of TLR7/8 agonist conjugates as a compelling strategy to combat opioid use disorders.

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Figures

Figure 1 Preparation and characterization of F1–CRM and F1–CRM-3006 conjugates. (a) Schematic illustration of preparation of the F1–CRM conjugate including chemical structure of F1 with a tetraglycine linker. (b) Schematic illustration of preparation of F1–CRM-3006 including synthesis and activation of UM-3006 with PEG linkers to 3006-PEG-NHS, the active NHS ester for conjugation. (c) Representative, normalized MALDI-ToF mass spectra of CRM, F1–CRM, and F1–CRM-3006. Rightmost peaks [M + H]+ and leftmost peaks [M + 2H]2+. (d) Image of a representative SDS-PAGE for nonreduced and reduced CRM (Lanes 2 and 3, respectively), F1–CRM (Lanes 4 and 5, respectively), and F1–CRM-3006 (Lanes 6 and 7, respectively). CRM structure obtained from PDB: 4AE0 and shown with lysine residues highlighted in blue.
Figure 1 Preparation and characterization of F1–CRM and F1–CRM-3006 conjugates. (a) Schematic illustration of preparation of the F1–CRM conjugate including chemical structure of F1 with a tetraglycine linker. (b) Schematic illustration of preparation of F1–CRM-3006 including synthesis and activation of UM-3006 with PEG linkers to 3006-PEG-NHS, the active NHS ester for conjugation. (c) Representative, normalized MALDI-ToF mass spectra of CRM, F1–CRM, and F1–CRM-3006. Rightmost peaks [M + H]+ and leftmost peaks [M + 2H]2+. (d) Image of a representative SDS-PAGE for nonreduced and reduced CRM (Lanes 2 and 3, respectively), F1–CRM (Lanes 4 and 5, respectively), and F1–CRM-3006 (Lanes 6 and 7, respectively). CRM structure obtained from PDB: 4AE0 and shown with lysine residues highlighted in blue.CC BY-NC-ND · PubMed Central
Figure 2 Vaccination of mice with coconjugates enhances F1-specific IgG antibody levels in sera. BALB/c mice (n = 6 or 8 per group) were immunized with 1 ug of CRM (1.06 μg F1–CRM) on days 0 and 14, and serum was harvested on day 28. Vaccines were adjuvanted with UM-3006 as coconjugates and 1× or 10× molar dose admixtures. Select groups also included alum. F1-specific serum (a) total IgG, (b) IgG2a, and (c) IgG1 titers were determined by ELISA on day 28. (d) Log of fold change in IgG2a and IgG1 to F1–CRM ratios to examine changes in Th1/Th2 polarization. Data are expressed as mean ± SEM. Brackets indicate select pairwise group comparisons using one-way ANOVA followed by Fisher’s LSD test. Symbols: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.
Figure 2 Vaccination of mice with coconjugates enhances F1-specific IgG antibody levels in sera. BALB/c mice (n = 6 or 8 per group) were immunized with 1 ug of CRM (1.06 μg F1–CRM) on days 0 and 14, and serum was harvested on day 28. Vaccines were adjuvanted with UM-3006 as coconjugates and 1× or 10× molar dose admixtures. Select groups also included alum. F1-specific serum (a) total IgG, (b) IgG2a, and (c) IgG1 titers were determined by ELISA on day 28. (d) Log of fold change in IgG2a and IgG1 to F1–CRM ratios to examine changes in Th1/Th2 polarization. Data are expressed as mean ± SEM. Brackets indicate select pairwise group comparisons using one-way ANOVA followed by Fisher’s LSD test. Symbols: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.CC BY-NC-ND · PubMed Central
Figure 3 Co-conjugation protects against the fentanyl challenge in mice. F1–CRM and F1–CRM-3006 conjugates were tested in BALB/c mice (n = 8 per group) to compare dose matched UM-3006 admix and coconjugate formulations with and without alum adjuvant. Injections of 1ug of F1–CRM were performed on days 0, 14, and 28 with 0.05 mg/kg sc fentanyl challenge on days (A–C) 21 and (D–F) 35. Data expressed as mean ± SEM. Brackets indicate pairwise comparisons using one-way ANOVA followed by Tukey’s multiple comparisons. Symbols: *p ≤ 0.05, **p ≤ 0.01, and ****p ≤ 0.0001.
Figure 3 Co-conjugation protects against the fentanyl challenge in mice. F1–CRM and F1–CRM-3006 conjugates were tested in BALB/c mice (n = 8 per group) to compare dose matched UM-3006 admix and coconjugate formulations with and without alum adjuvant. Injections of 1ug of F1–CRM were performed on days 0, 14, and 28 with 0.05 mg/kg sc fentanyl challenge on days (A–C) 21 and (D–F) 35. Data expressed as mean ± SEM. Brackets indicate pairwise comparisons using one-way ANOVA followed by Tukey’s multiple comparisons. Symbols: *p ≤ 0.05, **p ≤ 0.01, and ****p ≤ 0.0001.CC BY-NC-ND · PubMed Central
Figure 4 Vaccination with F1–CRM-3006 conjugates with or without alum increases serum IgG relative to the admixed control. Mice are the same as those shown in Figure 3. F1-specific IgG serum concentrations were determined on days (A) 14, (B) 28, and (C) 34. Data are expressed as mean ± SEM. Brackets indicate pairwise group comparisons using one-way ANOVA followed by the Fisher’s LSD test. Symbols: *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.
Figure 4 Vaccination with F1–CRM-3006 conjugates with or without alum increases serum IgG relative to the admixed control. Mice are the same as those shown in Figure 3. F1-specific IgG serum concentrations were determined on days (A) 14, (B) 28, and (C) 34. Data are expressed as mean ± SEM. Brackets indicate pairwise group comparisons using one-way ANOVA followed by the Fisher’s LSD test. Symbols: *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.CC BY-NC-ND · PubMed Central
Figure 5 Co-conjugate vaccination significantly restricts fentanyl distribution away from the brain relative to the admix control. Mice are the same as those shown in Figures 3 and 4 following the challenge on day 35. Fentanyl concentrations in the (A) serum and (B) brain were determined using LC-MS/MS. (C) Ratios of fentanyl in the serum and brain calculated for each mouse. Data are expressed as mean ± SEM. Brackets indicate pairwise comparisons using one-way ANOVA followed by Tukey’s multiple comparisons. Symbol: ****p ≤ 0.0001.
Figure 5 Co-conjugate vaccination significantly restricts fentanyl distribution away from the brain relative to the admix control. Mice are the same as those shown in Figures 3 and 4 following the challenge on day 35. Fentanyl concentrations in the (A) serum and (B) brain were determined using LC-MS/MS. (C) Ratios of fentanyl in the serum and brain calculated for each mouse. Data are expressed as mean ± SEM. Brackets indicate pairwise comparisons using one-way ANOVA followed by Tukey’s multiple comparisons. Symbol: ****p ≤ 0.0001.CC BY-NC-ND · PubMed Central

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Citations

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Metadata

Authors
Powers, N., Massena, C., Crouse, B., Smith, M., Hicks, L., Evans, J. T., Miller, S., Pravetoni, M., & Burkhart, D.
Affiliations
  • University of Montana (US)
  • University of Minnesota (US)
  • University of Washington (US)
Corresponding author
Burkhart, David
ORCID
Venue
Bioconjugate Chemistry
Publisher
American Chemical Society (ACS)
Published
2023-09-27
Volume
34
Issue
10
Pages
1811-1821
ISSN
  • 1043-1802 (ISSN-L)
  • 1520-4812
DOI
10.1021/acs.bioconjchem.3c00347
PMC
PMC10587865
OpenAlex
W4387079244
Cited by
15
References
4
Citations by year
2026: 2 · 2025: 6 · 2024: 6 · 2023: 1
FWCI
2.47
Type
journal article
Language
en
Open access
Licence
Locations
  • Bioconjugate Chemistry (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
Funders
  • National Institute on Drug Abuse (F31-DA054760, UG3DA048386)
  • National Institutes of Health (75N93020C00039, R01AI137146-01A1)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Monoclonal and Polyclonal Antibodies Research
  • Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
  • Health Sciences › Medicine › Public Health, Environmental and Occupational Health › Opioid Use Disorder Treatment
Keywords
fentanyl, opioid use disorder, TLR7/8 agonist, hapten conjugation, CRM197, aluminum hydroxide adjuvant, antibody titers, opioid overdose, vaccine adjuvants
MeSH terms
Adjuvants, Immunologic, Analgesics, Opioid, Animals, Antigens, Fentanyl, Haptens, Opioid-Related Disorders, Vaccines, Toll-Like Receptor 7, Mice

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